WO2020063667A1 - Solution changing method, solution changing plate, and uses thereof in cell staining and particle washing - Google Patents

Solution changing method, solution changing plate, and uses thereof in cell staining and particle washing Download PDF

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Publication number
WO2020063667A1
WO2020063667A1 PCT/CN2019/107804 CN2019107804W WO2020063667A1 WO 2020063667 A1 WO2020063667 A1 WO 2020063667A1 CN 2019107804 W CN2019107804 W CN 2019107804W WO 2020063667 A1 WO2020063667 A1 WO 2020063667A1
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WIPO (PCT)
Prior art keywords
tank
washing
liquid
buffer solution
sample
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PCT/CN2019/107804
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French (fr)
Chinese (zh)
Inventor
万瑛
王建
周见至
倪青山
邹丽云
李全滨
王晓玲
Original Assignee
重庆微浪生物科技有限公司
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Priority claimed from CN201821575639.2U external-priority patent/CN208999446U/en
Priority claimed from CN201811127633.3A external-priority patent/CN109270282A/en
Application filed by 重庆微浪生物科技有限公司 filed Critical 重庆微浪生物科技有限公司
Publication of WO2020063667A1 publication Critical patent/WO2020063667A1/en

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor

Definitions

  • the application belongs to the field of biotechnology, and particularly relates to a liquid exchange method, a liquid exchange plate, and uses thereof in cell staining and particle washing.
  • centrifugation to change the liquid.
  • the cell's activity, internal structure, and transcription mode will be affected to a certain extent. Impacts such as low cell recovery, high initial cell number requirements, adverse effects on cell status, unstable washing results, and difficulty in integrating with high-throughput automated sample preparation systems, so there are significant limitations to using centrifugal washing methods. For example, in flow cytometry, repeated centrifugation is required, resulting in poor cell status and loss of cells. Different operators will obtain different results.
  • the purpose of this application is to provide a liquid exchange method, a liquid exchange plate and its use in cell staining, to realize the non-centrifugal liquid exchange using the laws of gravity sedimentation and fluid diffusion, Able to treat cells gently and keep cells in good condition.
  • a liquid exchange plate provided in this application includes:
  • At least one liquid exchange unit disposed on the plate body, the liquid exchange unit includes a sample tank and a first wash tank and a second wash tank connected to both sides of the sample tank;
  • a separation protrusion is provided between the sample tank and the first washing liquid tank and between the sample tank and the second washing liquid tank, and the height of the separation protrusion is smaller than that of the first washing liquid tank and Depth of the second lotion tank.
  • the plate body is provided with a liquid exchange area, all the liquid exchange units are located in the liquid exchange area, and the plate body is provided with a cooling and moisturizing tank on the periphery of the liquid exchange area.
  • the top end of the sample tank communicates with the first and second lotion tanks through throats, respectively, and the width of the throats is smaller than the width and width of the first lotion tanks.
  • the width of the second washing liquid tank and the width of the sample tank are smaller than the width and width of the first lotion tanks.
  • every two adjacent liquid exchange units are arranged in parallel and staggered; in the horizontal direction, each The fluid exchange unit is on the same horizontal line.
  • the depth of the sample groove is 5-20 mm.
  • the height of the separation protrusion above the bottom surface of the sample slot is 1 mm-5 mm.
  • the specifications of the first washing liquid tank and the second washing liquid tank are the same.
  • a depth of the first washing liquid tank and the second washing liquid tank is 5 mm-20 mm.
  • the liquid exchange plate includes:
  • At least one liquid exchange unit disposed on the plate body, the liquid exchange unit includes a sample tank and a first wash tank and a second wash tank connected to both sides of the sample tank;
  • a separation protrusion is provided between the sample tank and the first washing liquid tank and between the sample tank and the second washing liquid tank, and the height of the separation protrusion is smaller than that of the first washing liquid tank and The depth of the second washing liquid tank;
  • the liquid exchange method further includes the following steps:
  • the speed of injecting the first buffer solution, the speed of aspiration of the first buffer solution, the speed of injecting the second buffer solution, and the aspiration of the second buffer solution in the liquid exchange method The speed is 0.01-10 ⁇ l / s.
  • the speed of injecting the first buffer solution in the liquid exchange method is the same as the speed of aspiration of the first buffer solution, and the speed of injecting the second buffer solution is as fast as the aspiration of the second buffer.
  • the speed of the liquid is the same.
  • the mixed cell suspension comprises a cell suspension added with a stain.
  • the volume of the mixed cell or particle suspension is 50-250 microliters.
  • the predetermined volume is 200-250 microliters.
  • the buffer solution, the first buffer solution and the second buffer solution are phosphate buffer solutions.
  • the purpose of the present application is also to provide the use of a liquid exchange method in cell staining and particle washing.
  • the liquid exchange method includes the following steps:
  • the liquid exchange plate includes:
  • At least one liquid exchange unit disposed on the plate body, the liquid exchange unit includes a sample tank and a first wash tank and a second wash tank connected to both sides of the sample tank;
  • a separation protrusion is provided between the sample tank and the first washing liquid tank and between the sample tank and the second washing liquid tank, and the height of the separation protrusion is smaller than that of the first washing liquid tank and The depth of the second washing liquid tank;
  • the cell staining includes intracellular cytokine staining or fluid surface antigen staining.
  • the step of staining the cells includes:
  • the staining result is analyzed based on the light signal intensity.
  • the stain includes a fluorescently labeled antibody.
  • the liquid exchange plate provided in the present application separates the bottom of the sample tank, the bottom of the first wash tank, and the bottom of the second wash tank in the liquid exchange unit through the separation protrusion, so that the sample cells can fully settle in the sample tank, and avoid the During the injection or drainage process, the sample cells that are precipitated will be disturbed to prevent the sample cells from being impacted and suspended or discharged as the cleaning solution flows.At the same time, the structure of the cleaning solution tank, the sample tank, and the separation protrusions cooperate to form a continuous and stable cleaning solution.
  • the flow channel enables the sample cells to be fully exchanged and cleaned, ensuring the processing effect and avoiding damage to the sample cells.
  • a liquid exchange method provided by the present application includes the steps of: injecting a mixed cell suspension or a particle suspension into at least one of the sample tanks on the liquid exchange plate, and allowing the mixed cell suspension to stand still The particles in the cell or particle suspension in the sedimentation settle; each of the first and second washing liquid tanks connected to the sample tank is filled with a buffer solution to a predetermined volume; and into the first washing liquid tank Injecting a first buffer solution and sucking out the first buffer solution from the second washing solution tank; injecting a second buffer solution into the second washing solution tank and sucking out the first washing solution tank Second buffer.
  • the present application utilizes the laws of gravity sedimentation and fluid diffusion to exchange liquids for cells, effectively improving the efficiency of cell liquid exchange.
  • the application also provides a use of a liquid exchange method in cell staining and particle washing, which utilizes the laws of gravity sedimentation and fluid diffusion to non-centrifugally stain specific substances.
  • the cells first settle in the buffer solution and slowly flow in the buffer solution. During the process, the cells basically remain stationary due to their inertia, which prevents a large number of cells in the liquid from being sucked away, reduces cell loss, and generates higher cell capture and sorting retention.
  • the buffer solution maintains a suitable flow rate in the sample tank, making the cells
  • the gentle washing can be performed continuously to avoid cell damage and shorten the workflow and time.
  • the number of cells retained by using the fluid exchange method provided by the present application is larger than that retained by the traditional centrifugation fluid exchange method, the detection result after the staining is stable, and the automation is convenient.
  • This application provides new staining protocols and tools for specific substance staining of cells.
  • FIG. 1 is a schematic flowchart of a method according to an embodiment of the present application
  • FIG. 2 is a schematic structural diagram of a liquid exchange plate in an embodiment of the present application.
  • FIG. 3 is a cross-sectional view of a liquid exchange unit in an embodiment of the present application.
  • FIG. 4 is a schematic diagram of the effect of different liquid exchange methods on the cell survival rate in an embodiment of the present application.
  • FIG. 5 is a schematic diagram of the effects of different fluid exchange methods on B lymphocytes of mice in an embodiment of the present application.
  • FIG. 6 is a schematic diagram of the effects of different fluid exchange methods on mouse T lymphocytes in an embodiment of the present application.
  • FIG. 7 is a schematic diagram of the effects of different fluid exchange methods on mouse CD4T lymphocytes in an embodiment of the present application.
  • FIG. 8 is a schematic diagram of the effects of different fluid exchange methods on mouse CD8T lymphocytes in an embodiment of the present application.
  • FIG. 9 shows the effects of different fluid exchange methods on mouse CD4T lymphocytes, mouse CD8T lymphocytes, mouse B lymphocytes, and natural killer cell staining indexes in an embodiment of the present application;
  • FIG. 10 is a schematic diagram of the effect of different fluid exchange methods on cell survival rate when staining for intracellular cytokines in an embodiment of the present application.
  • FIG. 11 shows the effect of different fluid exchange methods on the CD4T lymphocyte positive rate in mice according to an embodiment of the present application.
  • FIG. 12 is a schematic diagram showing the effects of different liquid exchange methods on the survival rate of living cells and dead cells in an embodiment of the present application.
  • FIG. 13 shows the effects of different fluid exchange methods on the positive staining rate of anti- ⁇ -interferon antibody labeled with phycoerythrin for mouse T lymphocytes and mouse CD4 T lymphocytes in an embodiment of the present application;
  • FIG. 14 shows the effects of different fluid exchange methods on the positive staining rate of anti- ⁇ -interferon antibodies labeled with phycoerythrin for mouse T lymphocytes and mouse CD8 T lymphocytes in an embodiment of the present application;
  • FIG. 15 is a schematic diagram of a cell staining process in an embodiment of the present application.
  • FIG. 1 is a liquid exchange method provided by an embodiment of the present application, including the following steps:
  • the liquid exchange plate includes a plate body 1, at least one liquid exchange unit 2, and a separation protrusion 23. At least one of the liquid exchange units 2 is disposed on the plate body 1.
  • the liquid exchange unit 2 includes a sample tank 21, and a first wash tank 22 and a second wash tank connected to both sides of the sample tank 21. 24; the separation protrusion 23 is provided between the sample tank 21 and the first washing liquid tank 22 and between the sample tank 21 and the second washing liquid tank 24, the separation protrusion 23
  • the height is smaller than the depth of the first washing liquid tank 22 and the second washing liquid tank 24, and is used to connect the sample tank 21 and the first washing liquid tank 22 and between the sample tank 21 and the second washing tank.
  • the liquid tanks 24 are spaced apart.
  • the height of the separation protrusion 23 refers to the height of the top of the separation protrusion 23 from the plane where the bottom of the first washing liquid tank 22 or the bottom of the second washing liquid tank 24 is located.
  • the depth of the second washing liquid tank 24 refers to the distance between the top of the first washing liquid tank 22 and the bottom of the first washing liquid tank 22 or between the top of the second washing liquid tank 24 and the bottom of the second washing liquid tank 24.
  • the upper part of the sample tank 21 communicates with the first washing liquid tank 22 and the second washing liquid tank 24 respectively.
  • the mixed cell suspension includes a cell suspension added with a stain.
  • the volume of the mixed cell suspension or particle suspension, ie, the sample, ranges from 50 to 250 ul.
  • the stain includes a fluorescently labeled antibody.
  • the buffer solution, the first buffer solution, and the second buffer solution are phosphate buffer solutions.
  • the predetermined volume is 200-250 microliters.
  • step S103 the speed of injecting the first buffer solution, the speed of aspiration of the first buffer solution, the speed of injecting the second buffer solution, and the aspiration of the second buffer in the liquid exchange method.
  • the speed of the buffer is 0.01-10 microliters / second.
  • the speed of injecting the first buffer solution is the same as the speed of aspiration of the first buffer solution
  • the speed of injecting the second buffer solution is the same as the speed of aspiration of the second buffer solution.
  • the volume of the first buffer solution drawn from the second washing solution tank 24 includes the same volume as the first buffer solution, and also includes a volume different from the first buffer solution.
  • the volume of the second buffer solution drawn from the first washing solution tank 22 includes the same volume as the second buffer solution, and also includes a volume different from the second buffer solution.
  • step S104 that is, after sucking the second buffer solution from the first washing solution tank 22, 200-250 micrometers are each sucked out of the first washing solution tank 22 and the second washing solution tank 24. Litres of waste liquid. Continue to inject the buffer solution into the first wash solution tank 22 and the second wash solution tank 24 and leave them to stand still, and repeat the liquid change according to the liquid change method.
  • the fluid exchange method described in this application is suitable for cell staining. Please refer to FIG. 15.
  • the steps of cell staining include:
  • the staining agent includes a fluorescently labeled antibody.
  • the mouse lymphocyte surface antigen staining is used as an example, and the flow cytometry technique is used.
  • the cells used are mouse spleen lymphocytes. After the mouse spleen lymphocytes are lysed by red blood cells, phosphate buffer solution is added. After re-suspending the PBS, use this application for subsequent operations, and finally test on the machine.
  • Antibodies for mice are, for example, phycoerythrin-labeled anti-natural killer cell antibodies, namely PE-anti-NK1.1, phycoerythrin-labeled anti-CD45T lymphocyte antibodies, such as PE-Cy7-anti-CD45, and allophycocyanin.
  • the anti-CD4T lymphocyte antibody is APC-anti-CD4
  • the anti-B220 cell antibody labeled with UV / visible region dye is BV510-anti-B220
  • the anti-interferon antibody is IFN- ⁇ antibody
  • the fluorescent dye is, for example, 4 ', 6-Difluorenyl-2-phenylindole is DAPI.
  • the staining steps are as follows:
  • Phosphate buffered saline PBS was added to mouse spleen lymphocytes to adjust the density to 1 ⁇ 10 5 -1 ⁇ 10 7 cells / ml to obtain a cell suspension.
  • the antibody was added at a volume ratio of 1: 180-200 to obtain a mixed cell suspension.
  • the fluorescent dye DAPI was added 8-10 minutes before the on-machine detection after the staining protocol was completed.
  • (c) For example, suck 100-200ul of PBS with a pipette tip, and inject 100-200ul of PBS into the second washing liquid tank 24 at a uniform rate of 0.01-10 microliters per second, and synchronize from the first washing liquid tank 22 Aspirate the same volume of waste as the buffer.
  • the liquid in the exchange plate was collected, and the volume was made up to 200 ⁇ l to be measured.
  • the cell antibody staining result was detected by flow cytometry.
  • the flow cytometer used the fluorescence signal intensity to analyze the staining result.
  • Figure 4 to Figure 9 show the results of cell antibody staining using the fluid exchange method of the present application.
  • A is the control group and B is the The liquid replacement method group, C is the group using the traditional centrifugal liquid replacement method.
  • the results shown in FIGS. 4-9 show that the dyeing results of this embodiment are stable and the signal-to-noise ratio is low.
  • the results of this example show that the application of the present application to flow detection technology can overcome the shortcomings of centrifugal flow staining, the method can retain a larger number of cells, and the staining result is more stable and convenient for automation.
  • the cells that have not undergone the liquid exchange and have the same other conditions as a control are recorded as group A, and the cell survival rates under the two liquid exchange methods are compared. Please refer to FIG. 4.
  • the results show that there is a significant difference in the effect of the two liquid exchange methods on the cell survival rate P ⁇ 0.05.
  • the liquid exchange method of the present application can save more live cells than the centrifugal liquid exchange method.
  • FIG. 5 to FIG. 8 Please refer to FIG. 5 to FIG. 8 to study the traditional centrifugal fluid exchange method and the fluid exchange method of the present application on different leukocyte populations such as CD4T lymphocytes, namely CD4 + T cells, CD8T lymphocytes, namely CD8 + T cells, B lymphocytes and natural killer cells. That is, the proportion of NK cells affects.
  • the results shown in FIGS. 5-8 show that the percentage of each cell population in the sample prepared by the liquid exchange method of the present application is not significantly different from the sample prepared by the traditional centrifugal liquid exchange method P> 0.05.
  • FIG. 9 Please refer to FIG. 9 to study the effect of different fluid exchange methods on the staining index of each fluorescent antibody.
  • the results show that in the samples prepared by the liquid exchange method of the present application, the staining index of each fluorescent antibody is significantly higher than that of the samples prepared by the traditional centrifugal liquid exchange method, P ⁇ 0.05, indicating that the liquid exchange method of the present application is helpful to improve the flow detection. Sensitivity and accuracy.
  • the centrifugal fluid exchange method is currently the most commonly used sample fluid exchange technology in flow cytometry, which is used to remove excess fluorescent antibodies in the system and reduce non-specific signal generation. Its disadvantages are low cell recovery, high initial cell number requirements, adverse effects on cell status, unstable fluid exchange effects, and difficulty in integrating with high-throughput automated sample preparation systems. Therefore, the use of centrifugal fluid exchange method for flow sample preparation has great limitations. From the above test results, it can be known that the liquid exchange method of the present application has obvious advantages over the centrifugal liquid exchange method in terms of cell recovery, cell viability, and sample detection sensitivity, and the advantages will be more obvious when the initial cell volume is small; In good condition. The detection result of the liquid exchange method of the present application has lower noise interference, higher sensitivity and accuracy, and is beneficial to subsequent data processing.
  • an example is intracellular cytokine staining, which is obtained from mouse spleen cells.
  • the mouse spleen cells were resuspended in complete cell culture medium, incubated at 35-37 ° C for 2-4 hours, the cells were collected, centrifuged at 500-600rpm for 4-5 minutes, and the cells were resuspended in PBS to make the cells in suspension Obtain a cell suspension, and adjust the cell density to 4 ⁇ 10 7 -4 ⁇ 10 8 cells / ml with PBS, divide the cell suspension into several 50-60 ⁇ l samples, and make the number of cells in each sample reach 2 ⁇ 10 5 -2 ⁇ 10 6 pieces.
  • Antibodies other than IFN- ⁇ such as PE-anti-NK1.1, PerCP-Cy5.5-anti-CD3, PE-Cy7-anti-CD45, APC-anti-CD4, fluorescein isothiocyanate-labeled antibody
  • Mouse CD8T lymphocyte antibodies namely FITC-anti-mouse-CD8 and BV510-anti-B220, are configured into two antibody premixes, and each sample is mixed with the antibody premix in equal volumes and transferred to at least one sample slot of the exchange plate. In 21, incubate at 3-4 ° C for 25-30 minutes.
  • the first washing liquid tank 22 and the second washing liquid tank 24 described below are washing liquid tanks connected to both ends of the sample tank 21.
  • a sampling pipette to suck 100-200ul 0.5mol / L glycine first, and then inject 100-200 ⁇ l 0.5mol / L glycine at a uniform rate into the first washing solution tank 22 at a rate of 0.01-10 microliters / second.
  • the second washing liquid tank 24 sucks out the same volume of liquid simultaneously; at a rate of 0.01-10 microliters per second, 100-200 ⁇ l 0.5 mol / L glycine is uniformly injected into the second washing liquid tank 24 from the first
  • the washing liquid tank 22 sucks out the same volume of liquid simultaneously; 100-200 ⁇ l of liquid is sucked out from the first washing liquid tank 22 and the second washing liquid tank 24, respectively.
  • FIG. 10 shows the results of dyeing using the liquid exchange method of the present application, and shows the effect on the experimental results in the case of using the present application.
  • the centrifugation method is more efficient.
  • FIG. 11 shows that the liquid exchange method of the present application is applied to the dyeing field, and the dyeing result is more stable than that obtained by the traditional centrifugal liquid exchange method.
  • FIG. 13 shows the effects of different fluid exchange methods on the positive staining rate of PE-anti-IFN- ⁇ antibodies for T lymphocytes and CD4 T lymphocytes.
  • the effect of different fluid exchange methods on the positive staining rate of PE-anti-IFN- ⁇ antibody the results show that the fluid exchange method of the present application has advantages over the traditional centrifugal fluid exchange method.
  • the sample Due to the shrinkage of the dead cells in the sample during flow cytometry, the sample has a lower forward scattering FSC. With reference to FIG. 12, it is shown that larger live cells can be better preserved than smaller dead cells during the fluid exchange process.
  • the fluid exchange method of the present application has a higher proportion of live cells than the traditional centrifugal fluid exchange method. .
  • This application utilizes the law of gravity sedimentation and fluid diffusion to non-centrifugally stain cell-specific substances.
  • the cells first settle in the buffer solution. During the slow flow of the buffer solution, the cells basically remain immobile due to their inertia. A large number of cells are aspirated, reducing cell loss, resulting in higher cell capture and sorting retention.
  • the buffer solution maintains a suitable flow rate in the sample tank, allowing cells to be washed gently and continuously, avoiding cell damage and shortening Workflow and time.
  • the experimental results show that the method of the present application retains more cells than the traditional method, the detection results are stable after staining, and are convenient for automation.
  • This application provides new staining protocols and tools for specific substance staining of cells.
  • the method of the present application is also applicable to washing of particle suspensions, such as protein particles, exosomal particles, and the like.
  • liquid exchange method of the present application can not only be implemented based on the liquid exchange plate described in this application, other liquid exchange plates can also implement the liquid exchange method of the present application.
  • a liquid exchange plate provided in the present application includes a plate body 1, at least one liquid exchange unit 2, and a separation protrusion 23. At least one of the liquid exchange units 2 is disposed on the plate body 1.
  • the liquid exchange unit 2 includes a sample tank 21, and a first wash tank 22 and a second wash tank connected to both sides of the sample tank 21. 24; the separation protrusion 23 is provided between the sample tank 21 and the first washing liquid tank 22 and between the sample tank 21 and the second washing liquid tank 24, the separation protrusion 23
  • the height is smaller than the depth of the first washing liquid tank 22 and the second washing liquid tank 24, and is used to connect the sample tank 21 and the first washing liquid tank 22 and between the sample tank 21 and the second washing tank.
  • the liquid tanks 24 are spaced apart.
  • the tops of the partitioning protrusions 23 are lower than the tops of the first and second washing liquid tanks 22 and 24, so that the tops of the sample tank 21, the first and second washing liquid tanks 24 and 24 form cleaning liquid.
  • the washing solution completes the washing of the cell or particle suspension, that is, the sample cells during the flowing process.
  • the upper part of the sample tank 21 communicates with the first and second washing liquid tanks 24 through throats 25, and the width of the throat 25 is smaller than the width of the first washing liquid tank 22, the The width of the second washing solution tank 24 and the width of the sample tank 21 are beneficial to buffer the washing solution through the throat 25, reduce the impact of the washing solution, and avoid disturbing the sample cells that are deposited.
  • the depth range of the sample tank 21 is 5-20 mm
  • the depth range of the first wash tank 22 and the second wash tank 24 is 5-20 mm
  • the height of the separation protrusion 23 above the bottom surface of the sample tank 21 is 1 mm-5 mm.
  • the specifications of the first washing liquid tank 22 and the second washing liquid tank 24 are, for example, the same specifications.
  • the plate body 1 is provided with a liquid exchange area. All the liquid exchange units 2 are located in the liquid exchange area.
  • the plate body is provided with a cooling and moisturizing tank 3 on the periphery of the liquid exchange area.
  • the number of the liquid exchange units 2 is greater than or equal to two, in the longitudinal direction, every two adjacent liquid exchange units 2 are arranged in parallel and staggered; in the horizontal direction, each of the liquid exchange units 2 2 On the same horizontal line, this arrangement makes the structural layout compact and saves space.
  • the structural design is adopted, which is beneficial to reduce
  • the accumulated liquid at the bottom of the first washing liquid tank 22 and the bottom of the second washing liquid tank 24 is convenient for sucking the washing liquid in the first washing liquid tank 22 and the second washing liquid tank 24, and the suction is cleaner.
  • the positions of the bottom of the first washing liquid tank 22 and the bottom of the second washing liquid tank 24 also include positions lower than the bottom of the sample tank 21, and when the bottom of the first washing liquid tank 22 and the bottom of the second washing liquid tank 24 are located When it is lower than the bottom of the sample tank 21, it is beneficial to avoid the liquid changer from touching the bottom of the first wash tank 22 and the bottom of the second wash tank 24 when injecting or sucking the cleaning solution.
  • the positions of the bottom of the first washing liquid tank 22 and the bottom of the second washing liquid tank 24 also include being flush with the bottom of the sample tank 21.
  • the liquid exchange plate provided in the present application isolates the sample tank 21, the first wash tank 22, and the second wash tank 24 in the liquid exchange unit 2 through the separation protrusion 23, so that, for example, cells or particles can be fully precipitated in the sample tank. And avoid interference with the precipitated cells or particles during the injection or drainage process, avoid the cells or particles from being impacted and suspended or discharged with the flow of the cleaning solution, and the structure of the washing solution tank, the sample tank, and the separation protrusions are matched Forming a channel for the continuous and stable flow of the cleaning liquid, so that the sample cells can be fully physically exchanged for cleaning, ensuring the processing effect and avoiding damage to the sample such as cells.

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Abstract

A solution changing method, a solution changing plate, and uses thereof in cell staining and particle washing. The solution changing method comprises the following steps: on a solution changing plate, injecting a mixed cell suspension or particle suspension into at least one sample tank (21) and allowing to stand still, thus allowing the cells in the mixed cell suspension or the particles in the particle suspension to settle; continuously injecting a buffer solution into a first cleaning solution tank (22) and a second cleaning solution tank (24) connected to the sample tank (21), and continuously discharging the buffer solution from the second cleaning solution tank (24) and the first cleaning solution tank (22).

Description

一种换液方法、换液板及其在细胞染色和颗粒洗涤中的用途Liquid exchange method, liquid exchange plate and use thereof in cell staining and particle washing 技术领域Technical field
本申请属于生物技术领域，具体涉及一种换液方法、换液板及其在细胞染色和颗粒洗涤中的用途。The application belongs to the field of biotechnology, and particularly relates to a liquid exchange method, a liquid exchange plate, and uses thereof in cell staining and particle washing.
背景技术Background technique
目前，人们常使用离心的方式进行换液，但是对例如细胞悬液或颗粒悬液进行离心操作时，受离心力和流体静压力的作用，细胞的活性、内部结构、转录模式等会受到一定的影响，如细胞回收率低、对初始细胞数量要求高、对细胞状态有不利影响、洗涤效果不稳定、与高通量自动化制样***难以整合，因此使用离心洗涤法存在很大的局限性。例如在流式细胞检测中，需要反复离心，导致细胞状态变差及细胞丢失，不同人员进行操作会得到不同的结果。At present, people often use centrifugation to change the liquid. However, when centrifuging a cell suspension or a particle suspension, for example, due to the effect of centrifugal force and hydrostatic pressure, the cell's activity, internal structure, and transcription mode will be affected to a certain extent. Impacts such as low cell recovery, high initial cell number requirements, adverse effects on cell status, unstable washing results, and difficulty in integrating with high-throughput automated sample preparation systems, so there are significant limitations to using centrifugal washing methods. For example, in flow cytometry, repeated centrifugation is required, resulting in poor cell status and loss of cells. Different operators will obtain different results.
发明内容Summary of the Invention
鉴于以上所述现有技术的缺点，本申请的目的在于提供一种换液方法、换液板及其在细胞染色中的用途，利用重力沉降和流体扩散的规律来实现非离心式换液，能够对细胞处理轻柔，使细胞保持良好的状态。In view of the shortcomings of the prior art described above, the purpose of this application is to provide a liquid exchange method, a liquid exchange plate and its use in cell staining, to realize the non-centrifugal liquid exchange using the laws of gravity sedimentation and fluid diffusion, Able to treat cells gently and keep cells in good condition.
为了实现上述目的或者其他目的，本申请是通过以下技术方案实现的：In order to achieve the above or other purposes, this application is achieved through the following technical solutions:
本申请提供的一种换液板，包括：A liquid exchange plate provided in this application includes:
板体；Board
至少一个换液单元，设置在所述板体上，所述换液单元包括样本槽和连接在所述样本槽两侧的第一洗液槽和第二洗液槽；及At least one liquid exchange unit, disposed on the plate body, the liquid exchange unit includes a sample tank and a first wash tank and a second wash tank connected to both sides of the sample tank; and
分隔凸起，设置在所述样本槽与所述第一洗液槽之间以及所述样本槽与所述第二洗液槽之间，所述分隔凸起的高度小于第一洗液槽和第二洗液槽的深度。A separation protrusion is provided between the sample tank and the first washing liquid tank and between the sample tank and the second washing liquid tank, and the height of the separation protrusion is smaller than that of the first washing liquid tank and Depth of the second lotion tank.
在一实施例中，所述板体上设有换液区，所有的换液单元位于换液区内， 所述板体上位于换液区***设置有冷却保湿槽。In one embodiment, the plate body is provided with a liquid exchange area, all the liquid exchange units are located in the liquid exchange area, and the plate body is provided with a cooling and moisturizing tank on the periphery of the liquid exchange area.
在一实施例中，所述样本槽的顶端分别与所述第一洗液槽和第二洗液槽通过喉口连通，所述喉口的宽度小于所述第一洗液槽的宽度、所述第二洗液槽的宽度及和所述样本槽的宽度。In one embodiment, the top end of the sample tank communicates with the first and second lotion tanks through throats, respectively, and the width of the throats is smaller than the width and width of the first lotion tanks. The width of the second washing liquid tank and the width of the sample tank.
在一实施例中，当所述换液单元的个数大于或等于两个时，在纵向方向上，每相邻两个所述换液单元平行且交错排列；在横向方向上，每个所述换液单元在同一水平线上。In an embodiment, when the number of the liquid exchange units is greater than or equal to two, in the longitudinal direction, every two adjacent liquid exchange units are arranged in parallel and staggered; in the horizontal direction, each The fluid exchange unit is on the same horizontal line.
在一实施例中，其中所述样本槽的深度为5mm-20mm。In an embodiment, the depth of the sample groove is 5-20 mm.
在一实施例中，所述分隔凸起高出所述样本槽底面的高度为1mm-5mm。In one embodiment, the height of the separation protrusion above the bottom surface of the sample slot is 1 mm-5 mm.
在一实施例中，其中所述第一洗液槽与所述第二洗液槽的规格相同。In an embodiment, the specifications of the first washing liquid tank and the second washing liquid tank are the same.
在一实施例中，其中所述第一洗液槽与所述第二洗液槽的深度为5mm-20mm。In an embodiment, a depth of the first washing liquid tank and the second washing liquid tank is 5 mm-20 mm.
本申请的目的还在于提供一种换液方法，包括以下步骤：The purpose of this application is also to provide a liquid exchange method, which includes the following steps:
提供换液板，所述换液板包括：Provide a liquid exchange plate, the liquid exchange plate includes:
板体；Board
至少一个换液单元，设置在所述板体上，所述换液单元包括样本槽和连接在所述样本槽两侧的第一洗液槽和第二洗液槽；及At least one liquid exchange unit, disposed on the plate body, the liquid exchange unit includes a sample tank and a first wash tank and a second wash tank connected to both sides of the sample tank; and
分隔凸起，设置在所述样本槽与所述第一洗液槽之间以及所述样本槽与所述第二洗液槽之间，所述分隔凸起的高度小于第一洗液槽和第二洗液槽的深度；A separation protrusion is provided between the sample tank and the first washing liquid tank and between the sample tank and the second washing liquid tank, and the height of the separation protrusion is smaller than that of the first washing liquid tank and The depth of the second washing liquid tank;
在所述换液板上，向至少一个所述样本槽中注入混合细胞悬液或颗粒悬液并静置，使所述混合细胞悬液中的细胞或颗粒悬液中的颗粒沉降；Injecting a mixed cell suspension or particle suspension into at least one of the sample tanks on the liquid exchange plate, and allowing the cells to settle in the mixed cell suspension or particles in the particle suspension;
向连接在所述样本槽两端的第一洗液槽和第二洗液槽中各注入缓冲液至预定体积；Inject a buffer solution into each of the first washing liquid tank and the second washing liquid tank connected to both ends of the sample tank to a predetermined volume;
向所述第一洗液槽中注入第一缓冲液并从所述第二洗液槽中吸出所述第一缓冲液；Injecting a first buffer solution into the first washing solution tank and sucking out the first buffer solution from the second washing solution tank;
向所述第二洗液槽中注入第二缓冲液并从所述第一洗液槽中吸出所述第二缓冲液。Inject a second buffer solution into the second washing solution tank and aspirate the second buffer solution from the first washing solution tank.
在一实施例中，所述换液方法还包括以下步骤：In one embodiment, the liquid exchange method further includes the following steps:
从所述第一洗液槽中吸出所述第二缓冲液后，从所述第一洗液槽和第二 洗液槽中各吸出200-250微升缓冲液。After aspirating the second buffer solution from the first washing solution tank, 200-250 microliters of buffer solution is aspirated from each of the first washing solution tank and the second washing solution tank.
在一实施例中，在所述换液方法中注入所述第一缓冲液的速度、吸出所述第一缓冲液的速度、注入所述第二缓冲液的速度以及吸出所述第二缓冲液的速度为0.01-10微升/秒。In one embodiment, the speed of injecting the first buffer solution, the speed of aspiration of the first buffer solution, the speed of injecting the second buffer solution, and the aspiration of the second buffer solution in the liquid exchange method The speed is 0.01-10 μl / s.
在一实施例中，在所述换液方法中注入所述第一缓冲液的速度与吸出所述第一缓冲液的速度相同，注入所述第二缓冲液的速度与吸出所述第二缓冲液的速度相同。In an embodiment, the speed of injecting the first buffer solution in the liquid exchange method is the same as the speed of aspiration of the first buffer solution, and the speed of injecting the second buffer solution is as fast as the aspiration of the second buffer. The speed of the liquid is the same.
在一实施例中，其中所述混合细胞悬液包括加入染色剂的细胞悬液。In one embodiment, the mixed cell suspension comprises a cell suspension added with a stain.
在一实施例中，其中所述混合细胞悬液或颗粒悬液的体积为50-250微升。In one embodiment, the volume of the mixed cell or particle suspension is 50-250 microliters.
在一实施例中，其中所述预定体积为200-250微升。In one embodiment, the predetermined volume is 200-250 microliters.
在一实施例中，其中所述缓冲液、第一缓冲液以及第二缓冲液为磷酸盐缓冲液。In one embodiment, the buffer solution, the first buffer solution and the second buffer solution are phosphate buffer solutions.
本申请的目的还在于提供一种换液方法在细胞染色和颗粒洗涤中的用途，所述换液方法，包括以下步骤：The purpose of the present application is also to provide the use of a liquid exchange method in cell staining and particle washing. The liquid exchange method includes the following steps:
提供换液板，所述换液板包括：Provide a liquid exchange plate, the liquid exchange plate includes:
板体；Board
至少一个换液单元，设置在所述板体上，所述换液单元包括样本槽和连接在所述样本槽两侧的第一洗液槽和第二洗液槽；及At least one liquid exchange unit, disposed on the plate body, the liquid exchange unit includes a sample tank and a first wash tank and a second wash tank connected to both sides of the sample tank; and
分隔凸起，设置在所述样本槽与所述第一洗液槽之间以及所述样本槽与所述第二洗液槽之间，所述分隔凸起的高度小于第一洗液槽和第二洗液槽的深度；A separation protrusion is provided between the sample tank and the first washing liquid tank and between the sample tank and the second washing liquid tank, and the height of the separation protrusion is smaller than that of the first washing liquid tank and The depth of the second washing liquid tank;
在所述换液板上，向至少一个所述样本槽中注入混合细胞悬液或颗粒悬液并静置，使所述混合细胞悬液中的细胞或颗粒悬液中的颗粒沉降；Injecting a mixed cell suspension or particle suspension into at least one of the sample tanks on the liquid exchange plate, and allowing the cells to settle in the mixed cell suspension or particles in the particle suspension;
向连接在所述样本槽两端的第一洗液槽和第二洗液槽中各注入缓冲液至预定体积；Inject a buffer solution into each of the first washing liquid tank and the second washing liquid tank connected to both ends of the sample tank to a predetermined volume;
向所述第一洗液槽中注入第一缓冲液并从所述第二洗液槽中吸出所述第一缓冲液；Injecting a first buffer solution into the first washing solution tank and sucking out the first buffer solution from the second washing solution tank;
向所述第二洗液槽中注入第二缓冲液并从所述第一洗液槽中吸出所述第二缓冲液。Inject a second buffer solution into the second washing solution tank and aspirate the second buffer solution from the first washing solution tank.
在一实施例中，所述细胞染色包括胞内细胞因子染色或流体表面抗原染色。In one embodiment, the cell staining includes intracellular cytokine staining or fluid surface antigen staining.
在一实施例中，所述细胞染色的步骤包括，In one embodiment, the step of staining the cells includes:
向细胞悬液中加入染色剂，获得所述混合细胞悬液；Adding a stain to the cell suspension to obtain the mixed cell suspension;
使用所述换液方法对所述混合细胞悬液进行洗涤，获得洗涤后的混合细胞悬液；Washing the mixed cell suspension using the liquid exchange method to obtain a washed mixed cell suspension;
检测所述洗涤后的混合细胞悬液中的光信号强度；Detecting the intensity of the light signal in the washed mixed cell suspension;
根据所述光信号强度，分析染色结果。The staining result is analyzed based on the light signal intensity.
在一实施例中，所述染色剂包括用荧光标记的抗体。In one embodiment, the stain includes a fluorescently labeled antibody.
本申请提供的换液板通过分隔凸起将换液单元内的样本槽底部、第一洗液槽底部和第二洗液槽底部隔断，使得样本细胞能够充分在样本槽了沉淀，并且避免在注液或排液过程中对沉淀的样本细胞产生干扰，避免样本细胞受到冲击悬浮或者随清洗液流动而被排出，同时洗液槽、样本槽、分隔凸起的结构配合形成供清洗液连续稳定流动的通道，使得样本细胞能够充分的进行物理换液清洗，保证了处理效果，避免损伤样本细胞。本申请提供的一种换液方法，包括以下步骤：在所述换液板上，向至少一个所述样本槽中注入混合细胞悬液或颗粒悬液并静置，使所述混合细胞悬液中的细胞或颗粒悬液中的颗粒沉降；向连接在所述样本槽两端的第一洗液槽和第二洗液槽中各注入缓冲液至预定体积；向所述第一洗液槽中注入第一缓冲液并从所述第二洗液槽中吸出所述第一缓冲液；向所述第二洗液槽中注入第二缓冲液并从所述第一洗液槽中吸出所述第二缓冲液。本申请利用重力沉降和流体扩散的规律来对细胞进行换液，有效提高细胞换液的效率。本申请还提供一种换液方法在细胞染色和颗粒洗涤中的用途，利用重力沉降和流体扩散的规律对特异性物质非离心式染色，细胞先在缓冲液中沉降，在缓冲液缓慢流动的过程中细胞由于其惯性基本保持不动，避免液体内的细胞被大量吸走，减少细胞损失，产生更高的细胞捕获和分选保留，缓冲液在样品槽中保持合适的流动速度，使得细胞能够连续不断的进行温和的洗涤，避免细胞损伤，缩短了工作流程和时间。使用本申请提供的换液方法细胞保留数量较使用传统的离心换液方法细胞保留数量更多，染色后检测结果稳定，并且便于自动化。本申请为对细胞进行特异性物质染色提供了新的染色方案和工具。The liquid exchange plate provided in the present application separates the bottom of the sample tank, the bottom of the first wash tank, and the bottom of the second wash tank in the liquid exchange unit through the separation protrusion, so that the sample cells can fully settle in the sample tank, and avoid the During the injection or drainage process, the sample cells that are precipitated will be disturbed to prevent the sample cells from being impacted and suspended or discharged as the cleaning solution flows.At the same time, the structure of the cleaning solution tank, the sample tank, and the separation protrusions cooperate to form a continuous and stable cleaning solution. The flow channel enables the sample cells to be fully exchanged and cleaned, ensuring the processing effect and avoiding damage to the sample cells. A liquid exchange method provided by the present application includes the steps of: injecting a mixed cell suspension or a particle suspension into at least one of the sample tanks on the liquid exchange plate, and allowing the mixed cell suspension to stand still The particles in the cell or particle suspension in the sedimentation settle; each of the first and second washing liquid tanks connected to the sample tank is filled with a buffer solution to a predetermined volume; and into the first washing liquid tank Injecting a first buffer solution and sucking out the first buffer solution from the second washing solution tank; injecting a second buffer solution into the second washing solution tank and sucking out the first washing solution tank Second buffer. The present application utilizes the laws of gravity sedimentation and fluid diffusion to exchange liquids for cells, effectively improving the efficiency of cell liquid exchange. The application also provides a use of a liquid exchange method in cell staining and particle washing, which utilizes the laws of gravity sedimentation and fluid diffusion to non-centrifugally stain specific substances. The cells first settle in the buffer solution and slowly flow in the buffer solution. During the process, the cells basically remain stationary due to their inertia, which prevents a large number of cells in the liquid from being sucked away, reduces cell loss, and generates higher cell capture and sorting retention. The buffer solution maintains a suitable flow rate in the sample tank, making the cells The gentle washing can be performed continuously to avoid cell damage and shorten the workflow and time. The number of cells retained by using the fluid exchange method provided by the present application is larger than that retained by the traditional centrifugation fluid exchange method, the detection result after the staining is stable, and the automation is convenient. This application provides new staining protocols and tools for specific substance staining of cells.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
为了更清楚地说明本申请具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本申请的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。In order to more clearly illustrate the specific implementations of the present application or the technical solutions in the prior art, the drawings used in the specific implementations or prior art descriptions will be briefly introduced below. Obviously, the appended The drawings are some implementations of the present application. For those of ordinary skill in the art, other drawings can be obtained according to these drawings without paying creative labor.
图1为本申请一实施例的方法流程示意图；FIG. 1 is a schematic flowchart of a method according to an embodiment of the present application;
图2为本申请一实施例中换液板的结构示意图；2 is a schematic structural diagram of a liquid exchange plate in an embodiment of the present application;
图3为本申请一实施例中换液单元的剖视图。FIG. 3 is a cross-sectional view of a liquid exchange unit in an embodiment of the present application.
图4为本申请一实施例中不同换液方式对细胞存活率的影响示意图；FIG. 4 is a schematic diagram of the effect of different liquid exchange methods on the cell survival rate in an embodiment of the present application; FIG.
图5为本申请一实施例中不同换液方式对小鼠B淋巴细胞的影响示意图；5 is a schematic diagram of the effects of different fluid exchange methods on B lymphocytes of mice in an embodiment of the present application;
图6为本申请一实施例中不同换液方式对小鼠T淋巴细胞的影响示意图；6 is a schematic diagram of the effects of different fluid exchange methods on mouse T lymphocytes in an embodiment of the present application;
图7为本申请一实施例中不同换液方式对小鼠CD4T淋巴细胞的影响示意图；7 is a schematic diagram of the effects of different fluid exchange methods on mouse CD4T lymphocytes in an embodiment of the present application;
图8为本申请一实施例中不同换液方式对小鼠CD8T淋巴细胞的影响示意图；8 is a schematic diagram of the effects of different fluid exchange methods on mouse CD8T lymphocytes in an embodiment of the present application;
图9为本申请一实施例中不同换液方式对小鼠CD4T淋巴细胞、小鼠CD8T淋巴细胞、小鼠B淋巴细胞和自然杀伤细胞染色指数的影响；FIG. 9 shows the effects of different fluid exchange methods on mouse CD4T lymphocytes, mouse CD8T lymphocytes, mouse B lymphocytes, and natural killer cell staining indexes in an embodiment of the present application;
图10为本申请一实施例中针对胞内细胞因子染色时不同换液方式对细胞存活率的影响示意图；FIG. 10 is a schematic diagram of the effect of different fluid exchange methods on cell survival rate when staining for intracellular cytokines in an embodiment of the present application; FIG.
图11为本申请一实施例中不同换液方式对小鼠CD4T淋巴细胞阳性率的影响；FIG. 11 shows the effect of different fluid exchange methods on the CD4T lymphocyte positive rate in mice according to an embodiment of the present application; FIG.
图12为本申请一实施例中不同换液方式对活细胞和死细胞存活率的影响示意图；FIG. 12 is a schematic diagram showing the effects of different liquid exchange methods on the survival rate of living cells and dead cells in an embodiment of the present application; FIG.
图13为本申请一实施例中针对小鼠T淋巴细胞和小鼠CD4T淋巴细胞，不同换液方式对用藻红蛋白标记的抗γ干扰素抗体阳性染色率的影响；FIG. 13 shows the effects of different fluid exchange methods on the positive staining rate of anti-γ-interferon antibody labeled with phycoerythrin for mouse T lymphocytes and mouse CD4 T lymphocytes in an embodiment of the present application;
图14为本申请一实施例中针对小鼠T淋巴细胞和小鼠CD8T淋巴细胞，不同换液方式对用藻红蛋白标记的抗γ干扰素抗体阳性染色率的影响；FIG. 14 shows the effects of different fluid exchange methods on the positive staining rate of anti-γ-interferon antibodies labeled with phycoerythrin for mouse T lymphocytes and mouse CD8 T lymphocytes in an embodiment of the present application;
图15为本申请一实施例中细胞染色的流程示意图。FIG. 15 is a schematic diagram of a cell staining process in an embodiment of the present application.
具体实施方式detailed description
下面将结合附图对本申请的技术方案进行清楚、完整地描述，显然，所描述的实施例是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。The technical solution of the present application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are a part of the embodiments of the present application, but not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative efforts shall fall within the protection scope of the present application.
在本申请的描述中，需要说明的是，如出现术语“中心”、“上”、“下”、“左”、“右”、“竖直”、“水平”、“内”、“外”等，其所指示的方位或位置关系为基于附图所示的方位或位置关系，仅是为了便于描述本申请和简化描述，而不是指示或暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作，因此不能理解为对本申请的限制。此外，如出现术语“第一”、“第二”、仅用于描述目的，而不能理解为指示或暗示相对重要性。其中，术语“第一位置”和“第二位置”为两个不同的位置。In the description of this application, it should be noted that if the terms "center", "up", "down", "left", "right", "vertical", "horizontal", "inside", "outside" appear ", Etc., the orientation or position relationship indicated is based on the orientation or position relationship shown in the drawings, and is only for the convenience of describing this application and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation It is constructed and operated in a specific orientation, so it cannot be understood as a limitation on this application. In addition, if the terms "first" and "second" appear, they are used for descriptive purposes only and should not be interpreted as indicating or implying relative importance. The terms “first position” and “second position” are two different positions.
在本申请的描述中，需要说明的是，除非另有明确的规定和限定，术语“安装”、“相连”、“连接”应做广义理解，例如，可以是固定连接，也可以是可拆卸连接，或一体地连接；可以是机械连接，也可以是电连接；可以是直接相连，也可以通过中间媒介间接相连，可以是两个元件内部的连通。对于本领域的普通技术人员而言，可以具体情况理解上述术语在本申请中的具体含义。In the description of this application, it should be noted that the terms "installation", "connected", and "connected" should be understood in a broad sense unless explicitly stated and limited otherwise. For example, they may be fixed connections or removable. Connected or integrated; it can be mechanical or electrical; it can be directly connected, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two elements. For those of ordinary skill in the art, the specific meanings of the above terms in this application can be understood in specific situations.
当实施例给出数值范围时，应理解，除非本申请另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本申请中使用的所有技术和科学术语与本技术领域的技术人员对现有技术的掌握及本申请的记载，还可以使用与本申请实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本申请。When the embodiment gives a numerical range, it should be understood that, unless otherwise specified in the present application, the two endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this application and those skilled in the art's grasp of the prior art and the description of this application can also use the methods, equipment and materials described in the examples of this application Any methods, equipment, and materials of similar or equivalent prior art to implement this application.
请参阅图1至图3，图1为本申请一个实施例提供的一种换液方法，包括如下步骤：Please refer to FIGS. 1 to 3. FIG. 1 is a liquid exchange method provided by an embodiment of the present application, including the following steps:
S100、提供换液板；S100. Provide liquid exchange plate;
S101、在所述换液板上，向至少一个所述样本槽21中注入混合细胞悬液或颗粒悬液并静置，使所述混合细胞悬液中的细胞或颗粒悬液中的颗粒沉降；S101. Injecting a mixed cell suspension or a particle suspension into at least one of the sample tanks 21 on the liquid exchange plate, and allowing the cells in the mixed cell suspension or the particles in the particle suspension to settle. ;
S102、向连接在所述样本槽21两端的第一洗液槽22和第二洗液槽24中各注入缓冲液至预定体积；S102. Inject a buffer solution into each of the first washing solution tank 22 and the second washing solution tank 24 connected to both ends of the sample tank 21 to a predetermined volume;
S103、向所述第一洗液槽22中注入第一缓冲液并从所述第二洗液槽24中吸出所述第一缓冲液；S103. Inject a first buffer solution into the first washing solution tank 22 and aspirate the first buffer solution from the second washing solution tank 24.
S104、向所述第二洗液槽24中注入第二缓冲液并从所述第一洗液槽22中吸出所述第二缓冲液。S104. Inject a second buffer solution into the second washing solution tank 24 and aspirate the second buffer solution from the first washing solution tank 22.
具体的，在S100步骤中，请参阅图2至图3，所述换液板包括板体1，至少一个换液单元2以及分隔凸起23。至少一个所述换液单元2设置在所述板体1上，所述换液单元2包括样本槽21和连接在所述样本槽21两侧的第一洗液槽22和第二洗液槽24；所述分隔凸起23设置在所述样本槽21与所述第一洗液槽22之间以及所述样本槽21与所述第二洗液槽24之间，所述分隔凸起23的高度小于第一洗液槽22和第二洗液槽24的深度，用于将所述样本槽21与所述第一洗液槽22之间以及所述样本槽21与所述第二洗液槽24之间隔开。所述分隔凸起23的高度指分隔凸起23的顶部距离所述第一洗液槽22底部或所述第二洗液槽24底部所在平面的高度，所述第一洗液槽22和第二洗液槽24的深度指第一洗液槽22顶部与所述第一洗液槽22底部之间的距离或者所述第二洗液槽24顶部与所述第二洗液槽24底部之间的距离，所述样本槽21的上部分别与所述第一洗液槽22和第二洗液槽24连通。Specifically, in step S100, please refer to FIG. 2 to FIG. 3, the liquid exchange plate includes a plate body 1, at least one liquid exchange unit 2, and a separation protrusion 23. At least one of the liquid exchange units 2 is disposed on the plate body 1. The liquid exchange unit 2 includes a sample tank 21, and a first wash tank 22 and a second wash tank connected to both sides of the sample tank 21. 24; the separation protrusion 23 is provided between the sample tank 21 and the first washing liquid tank 22 and between the sample tank 21 and the second washing liquid tank 24, the separation protrusion 23 The height is smaller than the depth of the first washing liquid tank 22 and the second washing liquid tank 24, and is used to connect the sample tank 21 and the first washing liquid tank 22 and between the sample tank 21 and the second washing tank. The liquid tanks 24 are spaced apart. The height of the separation protrusion 23 refers to the height of the top of the separation protrusion 23 from the plane where the bottom of the first washing liquid tank 22 or the bottom of the second washing liquid tank 24 is located. The depth of the second washing liquid tank 24 refers to the distance between the top of the first washing liquid tank 22 and the bottom of the first washing liquid tank 22 or between the top of the second washing liquid tank 24 and the bottom of the second washing liquid tank 24. The upper part of the sample tank 21 communicates with the first washing liquid tank 22 and the second washing liquid tank 24 respectively.
具体的，在S101步骤中，所述混合细胞悬液包括加入染色剂的细胞悬液。所述混合细胞悬液或颗粒悬液即样本的体积范围为50-250ul。所述染色剂包括用荧光标记的抗体。Specifically, in step S101, the mixed cell suspension includes a cell suspension added with a stain. The volume of the mixed cell suspension or particle suspension, ie, the sample, ranges from 50 to 250 ul. The stain includes a fluorescently labeled antibody.
具体的，在S102步骤中，其中所述缓冲液、第一缓冲液以及第二缓冲液为磷酸盐缓冲液。所述预定体积为200-250微升。Specifically, in step S102, the buffer solution, the first buffer solution, and the second buffer solution are phosphate buffer solutions. The predetermined volume is 200-250 microliters.
具体的，在S103步骤中，在所述换液方法中注入所述第一缓冲液的速度、吸出所述第一缓冲液的速度、注入所述第二缓冲液的速度以及吸出所述第二缓冲液的速度为0.01-10微升/秒。在所述换液方法中注入所述第一缓冲液的速度与吸出所述第一缓冲液的速度相同，注入所述第二缓冲液的速度与吸出所述第二缓冲液的速度相同。从所述第二洗液槽24中吸出所述第一缓冲液的体积包括与所述第一缓冲液等体积，也包括与所述第一缓冲液的体积不同。同理，从所述第一洗液槽22中吸出所述第二缓冲液的体积包括与所述第二缓冲液等体积，也包括与所述第二缓冲液的体积不同。Specifically, in step S103, the speed of injecting the first buffer solution, the speed of aspiration of the first buffer solution, the speed of injecting the second buffer solution, and the aspiration of the second buffer in the liquid exchange method. The speed of the buffer is 0.01-10 microliters / second. In the liquid exchange method, the speed of injecting the first buffer solution is the same as the speed of aspiration of the first buffer solution, and the speed of injecting the second buffer solution is the same as the speed of aspiration of the second buffer solution. The volume of the first buffer solution drawn from the second washing solution tank 24 includes the same volume as the first buffer solution, and also includes a volume different from the first buffer solution. Similarly, the volume of the second buffer solution drawn from the first washing solution tank 22 includes the same volume as the second buffer solution, and also includes a volume different from the second buffer solution.
具体的，在S104步骤后即在从所述第一洗液槽22吸出所述第二缓冲液 后，从所述第一洗液槽22和第二洗液槽24中各吸出200-250微升的废液。继续向所述第一洗液槽22和第二洗液槽24中分别注入所述缓冲液并静置，根据所述换液方法重复换液。Specifically, after step S104, that is, after sucking the second buffer solution from the first washing solution tank 22, 200-250 micrometers are each sucked out of the first washing solution tank 22 and the second washing solution tank 24. Litres of waste liquid. Continue to inject the buffer solution into the first wash solution tank 22 and the second wash solution tank 24 and leave them to stand still, and repeat the liquid change according to the liquid change method.
本申请所述的换液方法适用于细胞染色，请参阅图15，所述细胞染色的步骤包括：The fluid exchange method described in this application is suitable for cell staining. Please refer to FIG. 15. The steps of cell staining include:
S201、向细胞悬液中加入染色剂，获得所述混合细胞悬液；S201. Add a stain to the cell suspension to obtain the mixed cell suspension.
S202、使用所述换液方法对所述混合细胞悬液进行洗涤，获得洗涤后的混合细胞悬液；S202. Wash the mixed cell suspension by using the liquid exchange method to obtain a washed mixed cell suspension;
S203、检测所述洗涤后的混合细胞悬液中的光信号强度；S203: Detect the intensity of the light signal in the washed mixed cell suspension;
S204、根据所述光信号强度，分析染色结果。S204. Analyze the staining result according to the light signal intensity.
具体的，在S201步骤中，所述染色剂包括用荧光标记的抗体。Specifically, in step S201, the staining agent includes a fluorescently labeled antibody.
将本申请的换液方法应用于胞内细胞因子染色或流体表面抗原染色为例，具体说明本申请。The application of the fluid exchange method of the present application to intracellular cytokine staining or fluid surface antigen staining is taken as an example, and the present application is specifically described.
在一实施例中，以小鼠淋巴细胞表面抗原染色为例，采用的是流式染色技术，所用细胞为小鼠脾脏淋巴细胞，将小鼠脾脏淋巴细胞经过裂解红细胞过后，加磷酸盐缓冲液PBS重悬后使用本申请进行后续操作，并最终上机检测。In one embodiment, the mouse lymphocyte surface antigen staining is used as an example, and the flow cytometry technique is used. The cells used are mouse spleen lymphocytes. After the mouse spleen lymphocytes are lysed by red blood cells, phosphate buffer solution is added. After re-suspending the PBS, use this application for subsequent operations, and finally test on the machine.
小鼠用抗体例如为藻红蛋白标记的抗自然杀伤细胞抗体即PE-anti-NK1.1、藻红蛋白标记的抗CD45T淋巴细胞抗体即PE-Cy7-anti-CD45、别藻蓝蛋白标记的抗CD4T淋巴细胞抗体即APC-anti-CD4、用紫外/可见光区染料标记的抗B220细胞的抗体即BV510-anti-B220、抗γ干扰素抗体即IFN-γ抗体，荧光染料例如为4'，6-二脒基-2-苯基吲哚即DAPI。Antibodies for mice are, for example, phycoerythrin-labeled anti-natural killer cell antibodies, namely PE-anti-NK1.1, phycoerythrin-labeled anti-CD45T lymphocyte antibodies, such as PE-Cy7-anti-CD45, and allophycocyanin. The anti-CD4T lymphocyte antibody is APC-anti-CD4, the anti-B220 cell antibody labeled with UV / visible region dye is BV510-anti-B220, the anti-interferon antibody is IFN-γ antibody, and the fluorescent dye is, for example, 4 ', 6-Difluorenyl-2-phenylindole is DAPI.
染色步骤具体如下：The staining steps are as follows:
(1)对小鼠脾脏淋巴细胞进行孵育和染色(1) Incubation and staining of mouse spleen lymphocytes
向小鼠脾脏淋巴细胞中加入磷酸盐缓冲液PBS调密度至1×10 5-1×10 7个/ml，获得细胞悬液，取800-900ul所述细胞悬液，按细胞悬液和抗体的体积比为1:180-200加入抗体，获得混合细胞悬液，其中，对于荧光染料DAPI是在染色方案完成后，上机检测前8-10分钟加入。 Phosphate buffered saline PBS was added to mouse spleen lymphocytes to adjust the density to 1 × 10 5 -1 × 10 7 cells / ml to obtain a cell suspension. Take 800-900ul of the cell suspension, according to the cell suspension and antibodies. The antibody was added at a volume ratio of 1: 180-200 to obtain a mixed cell suspension. Among them, the fluorescent dye DAPI was added 8-10 minutes before the on-machine detection after the staining protocol was completed.
向换液板的每个样本槽中加入80-100μl的混合细胞悬液，使每孔有 10 5-10 6个细胞，静置30-40分钟，使所述细胞沉降。 Add 80-100 μl of mixed cell suspension to each sample well of the exchange plate, so that there are 10 5 -10 6 cells per well, and let stand for 30-40 minutes to allow the cells to settle.
(2)多余抗体去除(2) Excessive antibody removal
(a)在换液板的每个换液单元2中的第一洗液槽22和第二洗液槽24中各加入200-250μl磷酸盐缓冲液PBS(分2次加入)，静置3-5分钟，使所述细胞沉降。(a) Add 200-250 μl of phosphate buffered saline PBS (add 2 times) to each of the first and second wash tanks 22 and 24 in each exchange unit 2 of the exchange plate, and let stand 3 -5 minutes to allow the cells to settle.
(b)用例如加样枪头吸取100-200ul PBS，按0.01-10微升/秒的速度向所述第一洗液槽22中匀速注入100-200ul PBS，从第二洗液槽24中同步吸出与所述缓冲液等体积的废液。(b) Suction 100-200ul PBS with a pipette tip, for example, inject 100-200ul PBS into the first washing liquid tank 22 at a uniform rate at a rate of 0.01-10 microliters / second, and from the second washing liquid tank 24 Simultaneously aspirate the same volume of waste liquid as the buffer.
(c)用例如加样枪头吸取100-200ul PBS，0.01-10微升/秒的速度向所述第二洗液槽24中匀速注入100-200ul PBS，从第一洗液槽22中同步吸出与所述缓冲液等体积的废液。(c) For example, suck 100-200ul of PBS with a pipette tip, and inject 100-200ul of PBS into the second washing liquid tank 24 at a uniform rate of 0.01-10 microliters per second, and synchronize from the first washing liquid tank 22 Aspirate the same volume of waste as the buffer.
(d)从所述第一洗液槽22和第二洗液槽24中各吸出液体100-200μl，并丢弃。(d) Aspirate 100-200 μl of liquid from each of the first washing liquid tank 22 and the second washing liquid tank 24 and discard them.
(e)向所述第一洗液槽22和第二洗液槽24中各注入液体100-200μl，静置3-5分钟。(e) Inject 100-200 μl of liquid into each of the first washing liquid tank 22 and the second washing liquid tank 24, and let them stand for 3-5 minutes.
重复步骤(b)-(e)1次。Repeat steps (b)-(e) once.
重复步骤(b)-(d)1次。Repeat steps (b)-(d) once.
最后从所述第一洗液槽22和第二洗液槽24中同时吸出液体100-200μl。Finally, 100-200 μl of liquid is simultaneously aspirated from the first washing liquid tank 22 and the second washing liquid tank 24.
(3)染色完成并检测(3) Dyeing is completed and detected
收集换液板中的液体，体积补至200μl待测，用流式细胞仪检测细胞抗体染色结果，流式细胞仪是利用荧光信号强度来分析染色结果。The liquid in the exchange plate was collected, and the volume was made up to 200 μl to be measured. The cell antibody staining result was detected by flow cytometry. The flow cytometer used the fluorescence signal intensity to analyze the staining result.
检测结果请参阅图4-图9，图4-图9显示了采用本申请的换液方法进行细胞抗体染色的结果，在图4-图9中，A为对照组，B为运用本申请的换液方法组，C为运用传统的离心换液方法组，图4-图9结果显示出，本实施例染色结果稳定且信噪比低。本实施例的结果表明，将本申请应用于流式检测技术，能克服离心式流式染色的缺陷，本方法能保留更多数量的细胞，染色结果更稳定且便于自动化。Please refer to Figure 4 to Figure 9 for the test results. Figure 4 to Figure 9 show the results of cell antibody staining using the fluid exchange method of the present application. In Figure 4 to Figure 9, A is the control group and B is the The liquid replacement method group, C is the group using the traditional centrifugal liquid replacement method. The results shown in FIGS. 4-9 show that the dyeing results of this embodiment are stable and the signal-to-noise ratio is low. The results of this example show that the application of the present application to flow detection technology can overcome the shortcomings of centrifugal flow staining, the method can retain a larger number of cells, and the staining result is more stable and convenient for automation.
为了比较离心换液方法和本申请换液方法对细胞存活率的影响，以没有进行换液且其他条件相同的细胞为对照，记为A组，比较两种换液方式下细胞的存活率，请参阅图4，结果显示两种换液方式对细胞存活率的影响有显 著区别P<0.05，本申请的换液方法比离心换液方法能保存更多的活细胞。In order to compare the effect of the centrifugation method and the method of the present application on the cell survival rate, the cells that have not undergone the liquid exchange and have the same other conditions as a control are recorded as group A, and the cell survival rates under the two liquid exchange methods are compared. Please refer to FIG. 4. The results show that there is a significant difference in the effect of the two liquid exchange methods on the cell survival rate P <0.05. The liquid exchange method of the present application can save more live cells than the centrifugal liquid exchange method.
请参阅图5至图8，研究传统的离心换液方法和本申请换液方法对不同白细胞群体CD4T淋巴细胞即CD4 +T细胞、CD8T淋巴细胞即CD8 +T细胞，B淋巴细胞和自然杀伤细胞即NK细胞的比例影响。如图5-图8结果显示本申请换液方法制备的样本中的各细胞群体百分比与传统离心换液方法制备的样本均无显著差异P>0.05。 Please refer to FIG. 5 to FIG. 8 to study the traditional centrifugal fluid exchange method and the fluid exchange method of the present application on different leukocyte populations such as CD4T lymphocytes, namely CD4 + T cells, CD8T lymphocytes, namely CD8 + T cells, B lymphocytes and natural killer cells. That is, the proportion of NK cells affects. The results shown in FIGS. 5-8 show that the percentage of each cell population in the sample prepared by the liquid exchange method of the present application is not significantly different from the sample prepared by the traditional centrifugal liquid exchange method P> 0.05.
请参阅图9，研究不同换液方式对各荧光抗体染色指数(Staining Index)的影响。结果显示本申请换液方法制备的样本中，各荧光抗体的染色指数均显著高于传统的离心换液方法制备的样本P<0.05，说明本申请的换液方法有助于提升流式检测的灵敏度和准确性。Please refer to FIG. 9 to study the effect of different fluid exchange methods on the staining index of each fluorescent antibody. The results show that in the samples prepared by the liquid exchange method of the present application, the staining index of each fluorescent antibody is significantly higher than that of the samples prepared by the traditional centrifugal liquid exchange method, P <0.05, indicating that the liquid exchange method of the present application is helpful to improve the flow detection. Sensitivity and accuracy.
离心换液方法是目前流式染色中最常用的样本换液技术，用于去除体系中多余的荧光抗体，减少非特异信号产生。其不足之处在于细胞回收率低、对初始细胞数量要求高、对细胞状态有不利影响、换液效果不稳定、与高通量自动化制样***难以整合。因此使用离心换液方法进行流式样本制备存在很大的局限性。由以上检测结果可知本申请的换液方法在细胞回收率、细胞活性和样本检测灵敏度上较离心换液方法有明显优势，在初始细胞量较少的情况下优势会更明显；且能保证细胞处于较好的状态。本申请换液方法的检测结果有更低的杂信号干扰，有更高的灵敏度和准确性，有利于后续的数据处理。The centrifugal fluid exchange method is currently the most commonly used sample fluid exchange technology in flow cytometry, which is used to remove excess fluorescent antibodies in the system and reduce non-specific signal generation. Its disadvantages are low cell recovery, high initial cell number requirements, adverse effects on cell status, unstable fluid exchange effects, and difficulty in integrating with high-throughput automated sample preparation systems. Therefore, the use of centrifugal fluid exchange method for flow sample preparation has great limitations. From the above test results, it can be known that the liquid exchange method of the present application has obvious advantages over the centrifugal liquid exchange method in terms of cell recovery, cell viability, and sample detection sensitivity, and the advantages will be more obvious when the initial cell volume is small; In good condition. The detection result of the liquid exchange method of the present application has lower noise interference, higher sensitivity and accuracy, and is beneficial to subsequent data processing.
在另一个实施例中，以胞内细胞因子染色为例，所述胞内细胞因子从小鼠脾脏细胞中获得。In another embodiment, an example is intracellular cytokine staining, which is obtained from mouse spleen cells.
(I)细胞准备(I) Cell preparation
将小鼠脾脏细胞放入完全细胞培养基中重悬，35-37℃孵育2-4小时，收集细胞，以500-600rpm的转速离心4-5分钟，用PBS重悬细胞使细胞处于悬浮状态获得细胞悬液，并用PBS调整细胞密度至4×10 7-4×10 8个/ml，将细胞悬液分为若干个50-60μl的样本，使每个样本的细胞数目达到2×10 5-2×10 6个。 The mouse spleen cells were resuspended in complete cell culture medium, incubated at 35-37 ° C for 2-4 hours, the cells were collected, centrifuged at 500-600rpm for 4-5 minutes, and the cells were resuspended in PBS to make the cells in suspension Obtain a cell suspension, and adjust the cell density to 4 × 10 7 -4 × 10 8 cells / ml with PBS, divide the cell suspension into several 50-60 μl samples, and make the number of cells in each sample reach 2 × 10 5 -2 × 10 6 pieces.
(II)表面抗体孵育(II) Surface antibody incubation
将除IFN-γ外的抗体如PE-anti-NK1.1、PerCP-Cy5.5-anti-CD3、PE-Cy7-anti-CD45、APC-anti-CD4、异硫氰酸荧光素标记的抗小鼠CD8T淋 巴细胞抗体即FITC-anti-mouse-CD8、BV510-anti-B220分别配置成两份抗体预混液，每个样本与抗体预混液等体积混合，转入换液板的至少一个样本槽21中，3-4℃孵育25-30分钟。以下所述的第一洗液槽22与第二洗液槽24均为连接在所述样本槽21两端的洗液槽。Antibodies other than IFN-γ such as PE-anti-NK1.1, PerCP-Cy5.5-anti-CD3, PE-Cy7-anti-CD45, APC-anti-CD4, fluorescein isothiocyanate-labeled antibody Mouse CD8T lymphocyte antibodies, namely FITC-anti-mouse-CD8 and BV510-anti-B220, are configured into two antibody premixes, and each sample is mixed with the antibody premix in equal volumes and transferred to at least one sample slot of the exchange plate. In 21, incubate at 3-4 ° C for 25-30 minutes. The first washing liquid tank 22 and the second washing liquid tank 24 described below are washing liquid tanks connected to both ends of the sample tank 21.
(III)表面抗体染色后换液(III) Change fluid after surface antibody staining
(a 1)用例如加样枪头吸取100-200ul PBS，按0.01-10微升/秒的速度向所述第一洗液槽22中匀速注入100-200ul PBS，从第二洗液槽24中同步吸出与所述缓冲液等体积的废液。 (a 1 ) Absorb 100-200ul PBS with a pipette tip, inject 100-200ul PBS into the first washing solution tank 22 at a uniform rate at a rate of 0.01-10 microliters / second, and from the second washing solution tank 24 Aspirate the same volume of waste liquid as the buffer.
(b 1)用例如加样枪头吸取100-200ul PBS，按0.01-10微升/秒的速度向所述第二洗液槽24中匀速注入100-200ul PBS，从第一洗液槽22中同步吸出与所述缓冲液等体积的废液。 (b 1 ) Using, for example, a pipette tip to suck 100-200ul of PBS, and inject 100-200ul of PBS into the second washing solution tank 24 at a uniform rate at a rate of 0.01-10 microliters / second, and from the first washing solution tank 22 Aspirate the same volume of waste liquid as the buffer.
(c 1)从所述第一洗液槽22和所述第二洗液槽24中各吸出液体100-200μl并丢弃。 (c 1 ) Aspirate 100-200 μl of liquid from each of the first washing liquid tank 22 and the second washing liquid tank 24 and discard them.
(d 1)向所述第一洗液槽22和所述第二洗液槽24中各注入液体100-200μl。 (d 1 ) Inject 100-200 μl of liquid into each of the first washing liquid tank 22 and the second washing liquid tank 24.
(e 1)静置3-5分钟。 (e 1 ) Let stand for 3-5 minutes.
重复步骤(a 1)-(e 1)1次。 Repeat steps (a 1 )-(e 1 ) once.
重复步骤(a 1)-(c 1)1次。 Repeat steps (a 1 )-(c 1 ) once.
最后从第一洗液槽22和所述第二洗液槽24中同步吸出液体100-200μl。Finally, 100-200 μl of liquid is simultaneously aspirated from the first washing liquid tank 22 and the second washing liquid tank 24.
(IV)使样品固定通透(IV) Make the sample fixed and transparent
向所述第一洗液槽22和所述第二洗液槽24各注入100-200μl的固定破膜剂Fix/Perm，用例如加样枪头先吸取100-200ul固定破膜剂Fix/Perm，然后按0.01-10微升/秒的速度向所述第一洗液槽22内匀速注入100-200μl固定破膜剂Fix/Perm，从所述第二洗液槽24中同步吸出与所述固定破膜剂等体积的液体；按0.01-10微升/秒的速度向所述第二洗液槽24内匀速注入100-200μl固定破膜剂Fix/Perm，再从所述第一洗液槽22同步吸出等体积液体，静置5-10分钟。Inject 100-200 μl of fixed film-breaking agent Fix / Perm into each of the first and second liquid- washing tanks 22 and 24, and first draw 100-200ul of fixed film-breaking agent Fix / Perm with, for example, a pipette tip. Then, at a rate of 0.01-10 microliters per second, 100-200 μl of fixed film-breaking agent Fix / Perm is injected into the first washing liquid tank 22 at a uniform rate, and the second washing liquid tank 24 is simultaneously sucked out and the Fixed film breaking agent equal volume of liquid; 100-200 μl of fixed film breaking agent Fix / Perm is uniformly injected into the second washing liquid tank 24 at a rate of 0.01-10 microliters per second, and then from the first washing liquid The tank 22 simultaneously sucks out the same volume of liquid and stands still for 5-10 minutes.
(V)固定液中和(V) Fixing solution neutralization
用例如加样枪头先吸取100-200ul 0.5mol/L甘氨酸，然后按0.01-10微升/秒的速度向所述第一洗液槽22内匀速注入100-200μl 0.5mol/L甘氨酸， 从所述第二洗液槽24同步吸出等体积液体；按0.01-10微升/秒的速度向所述第二洗液槽24内匀速注入100-200μl 0.5mol/L甘氨酸，从所述第一洗液槽22同步吸出等体积液体；从所述第一洗液槽22和所述第二洗液槽24各吸出100-200μl液体。Use, for example, a sampling pipette to suck 100-200ul 0.5mol / L glycine first, and then inject 100-200μl 0.5mol / L glycine at a uniform rate into the first washing solution tank 22 at a rate of 0.01-10 microliters / second. The second washing liquid tank 24 sucks out the same volume of liquid simultaneously; at a rate of 0.01-10 microliters per second, 100-200 μl 0.5 mol / L glycine is uniformly injected into the second washing liquid tank 24 from the first The washing liquid tank 22 sucks out the same volume of liquid simultaneously; 100-200 μl of liquid is sucked out from the first washing liquid tank 22 and the second washing liquid tank 24, respectively.
(VI)固定液中和后换液(VI) Change the fluid after neutralization
向所述第一洗液槽22和所述第二洗液槽24各注入100-200μl的渗透液；用例如加样枪头先吸取100-200ul的渗透液，然后按0.01-10微升/秒的速度向所述第一洗液槽22内匀速注入100-200μl的渗透液，从所述第二洗液槽24同步吸出等体积液体；用加样枪头按0.01-10微升/秒的速度向所述第二洗液槽24内匀速注入100-200μl的渗透液，从所述第一洗液槽同步吸出等体积的液体；从所述第一洗液槽22和所述第二洗液槽24各吸出100-200μl的液体。Inject 100-200 μl of permeate into each of the first wash solution tank 22 and the second wash solution tank 24; use, for example, a pipette tip to aspirate 100-200 ul of permeate, and then press 0.01-10 μl / Inject 100-200 μl of permeate into the first washing liquid tank 22 at a uniform speed, and simultaneously aspirate an equal volume of liquid from the second washing liquid tank 24; use a sampling pipette tip at 0.01-10 microliters / second 100-200 μl of permeate is injected into the second washing liquid tank 24 at a uniform speed, and an equal volume of liquid is simultaneously sucked out from the first washing liquid tank; the first washing liquid tank 22 and the second Each of the washing liquid tanks 24 aspirates 100-200 μl of liquid.
(VII)胞内因子抗体染色孵育(VII) Incubation with intracellular factor antibody staining
加入用藻红蛋白标记的抗γ干扰素抗体即PE-anti-IFN-γ抗体到换液板在3-4℃孵育20-30分钟。Add phycoerythrin-labeled anti-interferon antibody, PE-anti-IFN-γ antibody, to the plate and incubate at 3-4 ° C for 20-30 minutes.
(VIII)胞内未结合抗体的换液(VIII) Exchange of intracellular unbound antibody
在换液板的至少一个换液单元2中的第一洗液槽22和第二洗液槽24中各加入200-250μl渗透液，静置3-5分钟；用加样枪头先吸取100-200ul渗透液，然后按0.01-10微升/秒的速度向所述第一洗液槽22内匀速注入100-200μl渗透液，再从所述第二洗液槽24同步吸出等体积液体。Add 200-250 μl of permeate to each of the first and second washing tanks 22 and 24 in at least one of the liquid exchange units 2 of the liquid exchange plate, and let stand for 3-5 minutes. -200ul permeate, and then inject 100-200μl permeate into the first washing solution tank 22 at a uniform speed at a rate of 0.01-10 microliters / second, and then simultaneously aspirate an equal volume of liquid from the second washing solution tank 24.
用加样枪头按0.01-10微升/秒的速度向所述第二洗液槽24内注入100-200μl渗透液，从所述第一洗液槽22同步吸出等体积液体；从所述第一洗液槽22和所述第二洗液槽24各吸出100-200μl液体。Inject a 100-200 μl permeate into the second washing liquid tank 24 at a rate of 0.01-10 microliters per second with a sample pipette tip, and simultaneously aspirate an equal volume of liquid from the first washing liquid tank 22; The first washing liquid tank 22 and the second washing liquid tank 24 each aspirate 100-200 μl of liquid.
从所述第一洗液槽22和所述第二洗液槽24各吸出液体100-200μl并丢弃。Aspirate 100-200 μl of liquid from each of the first washing solution tank 22 and the second washing solution tank 24 and discard them.
向所述第一洗液槽22和所述第二洗液槽24各注入100-200μl的渗透液。Inject 100-200 μl of permeate into each of the first washing solution tank 22 and the second washing solution tank 24.
静置3-5分钟。Let stand for 3-5 minutes.
重复步骤(a 1)-(e 1)1次。 Repeat steps (a 1 )-(e 1 ) once.
重复步骤(a 1)-(c 1)1次。 Repeat steps (a 1 )-(c 1 ) once.
最后从所述第一洗液槽22和所述第二洗液槽24中同步吸出液体100-200μl。Finally, 100-200 μl of liquid is simultaneously aspirated from the first washing liquid tank 22 and the second washing liquid tank 24.
(IX)染色完成并检测(IX) Staining is completed and detected
收集换液板中的液体，体积补至200μl待测，用流式细胞仪检测。结果请参阅图10，图10显示出采用本申请的换液方法进行染色的结果，并展示在使用本申请的情况下对实验结果的影响，结果显示采用本申请的换液方法染色效率比传统的离心换液方法染色效率更高。The liquid in the exchange plate was collected, and the volume was made up to 200 μl to be measured, and detected by flow cytometry. Please refer to FIG. 10 for the results. FIG. 10 shows the results of dyeing using the liquid exchange method of the present application, and shows the effect on the experimental results in the case of using the present application. The centrifugation method is more efficient.
请参阅图11，图11表明利用本申请的换液法应用于染色领域，染色结果比利用传统的离心换液方法得到的结果更稳定。Please refer to FIG. 11. FIG. 11 shows that the liquid exchange method of the present application is applied to the dyeing field, and the dyeing result is more stable than that obtained by the traditional centrifugal liquid exchange method.
请参阅图13-图14，图13为针对T淋巴细胞和CD4T淋巴细胞，不同换液方式对PE-anti-IFN-γ抗体阳性染色率色影响；图14为针对T淋巴细胞和CD8T淋巴细胞，不同换液方式对PE-anti-IFN-γ抗体阳性染色率色影响，结果显示本申请的换液方法较传统的离心换液方法有优势。Please refer to FIG. 13 to FIG. 14. FIG. 13 shows the effects of different fluid exchange methods on the positive staining rate of PE-anti-IFN-γ antibodies for T lymphocytes and CD4 T lymphocytes. The effect of different fluid exchange methods on the positive staining rate of PE-anti-IFN-γ antibody, the results show that the fluid exchange method of the present application has advantages over the traditional centrifugal fluid exchange method.
由于流式染色过程中样本的死细胞因外形皱缩而变小，具有较低的前向散射FSC。结合图12，表明在换液过程中，较大的活细胞比较小的死细胞能更好地保存下来，本申请的换液方法相较于传统的离心换液方法有更高的活细胞比例。Due to the shrinkage of the dead cells in the sample during flow cytometry, the sample has a lower forward scattering FSC. With reference to FIG. 12, it is shown that larger live cells can be better preserved than smaller dead cells during the fluid exchange process. The fluid exchange method of the present application has a higher proportion of live cells than the traditional centrifugal fluid exchange method. .
本申请利用重力沉降和流体扩散的规律来对细胞特异性物质非离心式染色，细胞先在缓冲液中沉降，在缓冲液缓慢流动的过程中细胞由于其惯性基本保持不动，避免液体内的细胞被大量吸走，减少细胞损失，产生更高的细胞捕获和分选保留，缓冲液在样品槽中保持合适的流动速度，使得细胞能够连续不断的进行温和的洗涤，避免细胞损伤，缩短了工作流程和时间。实验结果表明，本申请的方法细胞保留数量较传统方法更多，染色后检测结果稳定，并且便于自动化。本申请为对细胞进行特异性物质染色提供了新的染色方案和工具。本申请的方法也适用于颗粒悬液的洗涤，例如蛋白颗粒、外泌体颗粒等。This application utilizes the law of gravity sedimentation and fluid diffusion to non-centrifugally stain cell-specific substances. The cells first settle in the buffer solution. During the slow flow of the buffer solution, the cells basically remain immobile due to their inertia. A large number of cells are aspirated, reducing cell loss, resulting in higher cell capture and sorting retention. The buffer solution maintains a suitable flow rate in the sample tank, allowing cells to be washed gently and continuously, avoiding cell damage and shortening Workflow and time. The experimental results show that the method of the present application retains more cells than the traditional method, the detection results are stable after staining, and are convenient for automation. This application provides new staining protocols and tools for specific substance staining of cells. The method of the present application is also applicable to washing of particle suspensions, such as protein particles, exosomal particles, and the like.
本申请的换液方法不仅仅基于本申请所述的换液板才能实现，其它换液板也能实现本申请的换液方法。The liquid exchange method of the present application can not only be implemented based on the liquid exchange plate described in this application, other liquid exchange plates can also implement the liquid exchange method of the present application.
请参阅图2至图3，本申请提供的一种换液板，包括：板体1，至少一个换液单元2以及分隔凸起23。至少一个所述换液单元2设置在所述板体1上，所述换液单元2包括样本槽21和连接在所述样本槽21两侧的第一洗液槽22和第二洗液槽24；所述分隔凸起23设置在所述样本槽21与所述第一洗液槽 22之间以及所述样本槽21与所述第二洗液槽24之间，所述分隔凸起23的高度小于第一洗液槽22和第二洗液槽24的深度，用于将所述样本槽21与所述第一洗液槽22之间以及所述样本槽21与所述第二洗液槽24之间隔开。所述分隔凸起23的顶部低于第一洗液槽22和第二洗液槽24的顶部，使得样本槽21、第一洗液槽22和第二洗液槽24的顶部形成供清洗液流动的通道，清洗液在流动过程中完成细胞或颗粒悬液即样本细胞的清洗。所述样本槽21的上部分别与所述第一洗液槽和第二洗液槽24通过喉口25连通，所述喉口25的宽度小于所述第一洗液槽22的宽度、所述第二洗液槽24的宽度及所述样本槽21的宽度，通过喉口25有利于对清洗液进行缓冲，减少清洗液的冲击，避免干扰沉淀的样本细胞。样本槽21的深度范围为5-20mm，第一洗液槽22和第二洗液槽24深度范围为5-20mm，分隔凸起23高出所述样本槽21底面的高度为1mm-5mm。第一洗液槽22和第二洗液槽24的规格例如为相同规格。Please refer to FIGS. 2 to 3. A liquid exchange plate provided in the present application includes a plate body 1, at least one liquid exchange unit 2, and a separation protrusion 23. At least one of the liquid exchange units 2 is disposed on the plate body 1. The liquid exchange unit 2 includes a sample tank 21, and a first wash tank 22 and a second wash tank connected to both sides of the sample tank 21. 24; the separation protrusion 23 is provided between the sample tank 21 and the first washing liquid tank 22 and between the sample tank 21 and the second washing liquid tank 24, the separation protrusion 23 The height is smaller than the depth of the first washing liquid tank 22 and the second washing liquid tank 24, and is used to connect the sample tank 21 and the first washing liquid tank 22 and between the sample tank 21 and the second washing tank. The liquid tanks 24 are spaced apart. The tops of the partitioning protrusions 23 are lower than the tops of the first and second washing liquid tanks 22 and 24, so that the tops of the sample tank 21, the first and second washing liquid tanks 24 and 24 form cleaning liquid. In the flow channel, the washing solution completes the washing of the cell or particle suspension, that is, the sample cells during the flowing process. The upper part of the sample tank 21 communicates with the first and second washing liquid tanks 24 through throats 25, and the width of the throat 25 is smaller than the width of the first washing liquid tank 22, the The width of the second washing solution tank 24 and the width of the sample tank 21 are beneficial to buffer the washing solution through the throat 25, reduce the impact of the washing solution, and avoid disturbing the sample cells that are deposited. The depth range of the sample tank 21 is 5-20 mm, the depth range of the first wash tank 22 and the second wash tank 24 is 5-20 mm, and the height of the separation protrusion 23 above the bottom surface of the sample tank 21 is 1 mm-5 mm. The specifications of the first washing liquid tank 22 and the second washing liquid tank 24 are, for example, the same specifications.
请参阅图2，所述板体1上设有换液区，所有的换液单元2位于换液区内，所述板体上位于换液区***设置有冷却保湿槽3。当所述换液单元2的个数大于或等于两个时，在纵向方向上，每相邻两个所述换液单元2平行且交错排列；在横向方向上，每个所述换液单元2在同一水平线上，这样排列使得结构布局紧凑，节省空间。Referring to FIG. 2, the plate body 1 is provided with a liquid exchange area. All the liquid exchange units 2 are located in the liquid exchange area. The plate body is provided with a cooling and moisturizing tank 3 on the periphery of the liquid exchange area. When the number of the liquid exchange units 2 is greater than or equal to two, in the longitudinal direction, every two adjacent liquid exchange units 2 are arranged in parallel and staggered; in the horizontal direction, each of the liquid exchange units 2 2 On the same horizontal line, this arrangement makes the structural layout compact and saves space.
请参阅图3，本申请提供的换液板，当第一洗液槽22的底部与第二洗液槽24的底部的位置高于样本槽21的底部时，采用该结构设计，有利于减少第一洗液槽22的底部与第二洗液槽24底部的积液，便于吸取第一洗液槽22与第二洗液槽24内的清洗液，吸取更加干净。另外，第一洗液槽22的底部与第二洗液槽24的底部的位置还包括低于样本槽21的底部，当第一洗液槽22的底部与第二洗液槽24的底部位置低于样本槽21的底部时，有利于避免换液器在注入或吸取清洗液时触碰第一洗液槽22的底部与第二洗液槽24的底部。第一洗液槽22的底部与第二洗液槽24的底部的位置还包括与样本槽21的底部齐平。Referring to FIG. 3, when the positions of the bottom of the first washing liquid tank 22 and the second washing liquid tank 24 are higher than the bottom of the sample tank 21 in the liquid exchange plate provided in the present application, the structural design is adopted, which is beneficial to reduce The accumulated liquid at the bottom of the first washing liquid tank 22 and the bottom of the second washing liquid tank 24 is convenient for sucking the washing liquid in the first washing liquid tank 22 and the second washing liquid tank 24, and the suction is cleaner. In addition, the positions of the bottom of the first washing liquid tank 22 and the bottom of the second washing liquid tank 24 also include positions lower than the bottom of the sample tank 21, and when the bottom of the first washing liquid tank 22 and the bottom of the second washing liquid tank 24 are located When it is lower than the bottom of the sample tank 21, it is beneficial to avoid the liquid changer from touching the bottom of the first wash tank 22 and the bottom of the second wash tank 24 when injecting or sucking the cleaning solution. The positions of the bottom of the first washing liquid tank 22 and the bottom of the second washing liquid tank 24 also include being flush with the bottom of the sample tank 21.
本申请提供的换液板通过分隔凸起23将换液单元2内的样本槽21、第一洗液槽22和第二洗液槽24隔断，使得例如细胞或颗粒能够充分在样本槽中沉淀，并且避免在注液或排液过程中对沉淀的细胞或颗粒产生干扰，避免 细胞或颗粒受到冲击悬浮或者随清洗液流动而被排出，同时洗液槽、样本槽、分隔凸起的结构配合形成供清洗液连续稳定流动的通道，使得样本细胞能够充分的进行物理换液清洗，保证了处理效果，避免损伤样本例如细胞。The liquid exchange plate provided in the present application isolates the sample tank 21, the first wash tank 22, and the second wash tank 24 in the liquid exchange unit 2 through the separation protrusion 23, so that, for example, cells or particles can be fully precipitated in the sample tank. And avoid interference with the precipitated cells or particles during the injection or drainage process, avoid the cells or particles from being impacted and suspended or discharged with the flow of the cleaning solution, and the structure of the washing solution tank, the sample tank, and the separation protrusions are matched Forming a channel for the continuous and stable flow of the cleaning liquid, so that the sample cells can be fully physically exchanged for cleaning, ensuring the processing effect and avoiding damage to the sample such as cells.
此外应理解，本申请中提到的一个或多个方法步骤并不排斥在所述组合步骤前后还可以存在其他方法步骤或在这些明确提到的步骤之间还可以***其他方法步骤，除非另有说明；还应理解，除非另有说明，各方法步骤的编号仅为鉴别各方法步骤的便利工具，而非为限制各方法步骤的排列次序或限定本申请可实施的范围，其相对关系的改变或调整，在无实质变更技术内容的情况下，当亦视为本申请可实施的范畴。In addition, it should be understood that one or more method steps mentioned in this application are not exclusive of other method steps that may exist before or after the combined steps or that other method steps may be inserted between these explicitly mentioned steps, unless otherwise There are explanations; it should also be understood that, unless otherwise stated, the numbering of each method step is only a convenient tool for identifying each method step, and is not intended to limit the order of each method step or to limit the scope of implementation of this application. Changes or adjustments, without substantial changes in technical content, shall also be deemed to be the scope of this application.
最后应说明的是：以上各实施例仅用以说明本申请的技术方案，而非对其限制；尽管参照前述各实施例对本申请进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本申请各实施例技术方案的范围。Finally, it should be noted that the above embodiments are only used to describe the technical solution of the present application, rather than limiting it. Although the present application is described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: The technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features are equivalently replaced; and these modifications or replacements do not deviate the essence of the corresponding technical solutions from the technical solutions of the embodiments of this application. range.

Claims (20)

  1. 一种换液板，包括：A liquid exchange plate includes:
    板体；Board
    至少一个换液单元，设置在所述板体上，所述换液单元包括样本槽和连接在所述样本槽两侧的第一洗液槽和第二洗液槽；及At least one liquid exchange unit, disposed on the plate body, the liquid exchange unit includes a sample tank and a first wash tank and a second wash tank connected to both sides of the sample tank; and
    分隔凸起，设置在所述样本槽与所述第一洗液槽之间以及所述样本槽与所述第二洗液槽之间，所述分隔凸起的高度小于第一洗液槽和第二洗液槽的深度。A separation protrusion is provided between the sample tank and the first washing liquid tank and between the sample tank and the second washing liquid tank, and the height of the separation protrusion is smaller than that of the first washing liquid tank and Depth of the second lotion tank.
  2. 根据权利要求1所述的一种换液板，所述板体上设有换液区，所有的换液单元位于换液区内，所述板体上位于换液区***设置有冷却保湿槽。The liquid exchange plate according to claim 1, wherein the plate body is provided with a liquid exchange area, all the liquid exchange units are located in the liquid exchange area, and the plate body is provided with a cooling and moisturizing tank on the periphery of the liquid exchange area. .
  3. 根据权利要求1所述的一种换液板，所述样本槽的顶端分别与所述第一洗液槽和第二洗液槽通过喉口连通，所述喉口的宽度小于所述第一洗液槽的宽度、所述第二洗液槽的宽度及和所述样本槽的宽度。The liquid exchange plate according to claim 1, wherein the top end of the sample tank communicates with the first and second washing tanks through throats, respectively, and the width of the throats is smaller than that of the first The width of the washing liquid tank, the width of the second washing liquid tank, and the width of the sample tank.
  4. 根据权利要求1所述的一种换液板，当所述换液单元的个数大于或等于两个时，在纵向方向上，每相邻两个所述换液单元平行且交错排列；在横向方向上，每个所述换液单元在同一水平线上。The liquid exchange plate according to claim 1, when the number of the liquid exchange units is greater than or equal to two, in a longitudinal direction, every two adjacent liquid exchange units are arranged in parallel and staggered; In the lateral direction, each of the liquid exchange units is on the same horizontal line.
  5. 根据权利要求1所述的一种换液板，其中所述样本槽的深度为5mm-20mm。The liquid exchange plate according to claim 1, wherein a depth of the sample groove is 5-20 mm.
  6. 根据权利要求1所述的一种换液板，所述分隔凸起高出所述样本槽底面的高度为1mm-5mm。The liquid exchange plate according to claim 1, wherein a height of the separation protrusion from a bottom surface of the sample groove is 1 mm to 5 mm.
  7. 根据权利要求1所述的一种换液板，其中所述第一洗液槽与所述第二洗液槽的规格相同。The liquid exchange plate according to claim 1, wherein the specifications of the first washing liquid tank and the second washing liquid tank are the same.
  8. 根据权利要求1所述的换液板，其中所述第一洗液槽和所述第二洗液槽的深度为5mm-20mm。The liquid exchange plate according to claim 1, wherein a depth of the first washing liquid tank and the second washing liquid tank is 5 mm-20 mm.
  9. 一种换液方法，包括以下步骤：A fluid changing method includes the following steps:
    提供换液板，所述换液板包括：Provide a liquid exchange plate, the liquid exchange plate includes:
    板体；Board
    至少一个换液单元，设置在所述板体上，所述换液单元包括样本槽和连接在所述样本槽两侧的第一洗液槽和第二洗液槽；及At least one liquid exchange unit, disposed on the plate body, the liquid exchange unit includes a sample tank and a first wash tank and a second wash tank connected to both sides of the sample tank; and
    分隔凸起，设置在所述样本槽与所述第一洗液槽之间以及所述样本槽与所述第二洗液槽之间，所述分隔凸起的高度小于第一洗液槽和第二洗液槽的深度；A separation protrusion is provided between the sample tank and the first washing liquid tank and between the sample tank and the second washing liquid tank, and the height of the separation protrusion is smaller than that of the first washing liquid tank and The depth of the second washing liquid tank;
    在所述换液板上，向至少一个所述样本槽中注入混合细胞悬液或颗粒悬液并静置，使所述混合细胞悬液中的细胞或颗粒悬液中的颗粒沉降；Injecting a mixed cell suspension or particle suspension into at least one of the sample tanks on the liquid exchange plate, and allowing the cells to settle in the mixed cell suspension or particles in the particle suspension;
    向连接在所述样本槽两端的第一洗液槽和第二洗液槽中各注入缓冲液至预定体积；Inject a buffer solution into each of the first washing liquid tank and the second washing liquid tank connected to both ends of the sample tank to a predetermined volume;
    向所述第一洗液槽中注入第一缓冲液并从所述第二洗液槽中吸出所述第一缓冲液；Injecting a first buffer solution into the first washing solution tank and sucking out the first buffer solution from the second washing solution tank;
    向所述第二洗液槽中注入第二缓冲液并从所述第一洗液槽中吸出所述第二缓冲液。Inject a second buffer solution into the second washing solution tank and aspirate the second buffer solution from the first washing solution tank.
  10. 根据权利要求9所述的一种换液方法，所述换液方法还包括以下步骤：The liquid exchange method according to claim 9, further comprising the following steps:
    在从所述第一洗液槽吸出与所述第二缓冲液后，从所述第一洗液槽和第二洗液槽中各吸出200-250微升缓冲液。After aspirating from the first washing liquid tank and the second buffer solution, 200-250 microliters of buffer solution is aspirated from each of the first washing liquid tank and the second washing liquid tank.
  11. 根据权利要求9所述的一种换液方法，在所述换液方法中注入所述第一缓冲液的速度、吸出所述第一缓冲液的速度、注入所述第二缓冲液的速度以及吸出所述第二缓冲液的速度为0.01-10微升/秒。The liquid exchange method according to claim 9, wherein a speed at which the first buffer solution is injected, a speed at which the first buffer solution is drawn out, a speed at which the second buffer solution is injected, and The second buffer is aspirated at a rate of 0.01-10 microliters / second.
  12. 根据权利要求9所述的一种换液方法，在所述换液方法中注入所述第一缓冲液的速度与吸出所述第一缓冲液的速度相同，注入所述第二缓冲液的速度与吸出所述第二缓冲液的速度相同。The liquid exchange method according to claim 9, wherein the speed of injecting the first buffer solution is the same as the speed of aspiration of the first buffer solution, and the speed of injecting the second buffer solution in the liquid exchange method. Same speed as the second buffer was aspirated.
  13. 根据权利要求9所述的一种换液方法，其中所述混合细胞悬液包括加入染色剂的细胞悬液。The method of claim 9, wherein the mixed cell suspension comprises a cell suspension added with a staining agent.
  14. 根据权利要求9所述的一种换液方法，其中所述混合细胞悬液或颗粒悬液的体积为50-250微升。The method according to claim 9, wherein the volume of the mixed cell or particle suspension is 50-250 microliters.
  15. 根据权利要求9所述的一种换液方法，其中所述预定体积为200-250微升。A fluid exchange method according to claim 9, wherein said predetermined volume is 200-250 microliters.
  16. 根据权利要求9所述的一种换液方法，其中所述缓冲液、第一缓冲液以及第二缓冲液为磷酸盐缓冲液。The method according to claim 9, wherein the buffer solution, the first buffer solution and the second buffer solution are phosphate buffer solutions.
  17. 一种换液方法在细胞染色和颗粒洗涤中的用途，所述换液方法包括 以下步骤：Use of a fluid exchange method in cell staining and particle washing, the fluid exchange method includes the following steps:
    提供换液板，所述换液板包括：Provide a liquid exchange plate, the liquid exchange plate includes:
    板体；Board
    至少一个换液单元，设置在所述板体上，所述换液单元包括样本槽和连接在所述样本槽两侧的第一洗液槽和第二洗液槽；及At least one liquid exchange unit, disposed on the plate body, the liquid exchange unit includes a sample tank and a first wash tank and a second wash tank connected to both sides of the sample tank; and
    分隔凸起，设置在所述样本槽与所述第一洗液槽之间以及所述样本槽与所述第二洗液槽之间，所述分隔凸起的高度小于第一洗液槽和第二洗液槽的深度；A separation protrusion is provided between the sample tank and the first washing liquid tank and between the sample tank and the second washing liquid tank, and the height of the separation protrusion is smaller than that of the first washing liquid tank and The depth of the second washing liquid tank;
    在所述换液板上，向至少一个所述样本槽中注入混合细胞悬液或颗粒悬液并静置，使所述混合细胞悬液中的细胞或颗粒悬液中的颗粒沉降；Injecting a mixed cell suspension or particle suspension into at least one of the sample tanks on the liquid exchange plate, and allowing the cells to settle in the mixed cell suspension or particles in the particle suspension;
    向连接在所述样本槽两端的第一洗液槽和第二洗液槽中各注入缓冲液至预定体积；Inject a buffer solution into each of the first washing liquid tank and the second washing liquid tank connected to both ends of the sample tank to a predetermined volume;
    向所述第一洗液槽中注入第一缓冲液并从所述第二洗液槽中吸出所述第一缓冲液；Injecting a first buffer solution into the first washing solution tank and sucking out the first buffer solution from the second washing solution tank;
    向所述第二洗液槽中注入第二缓冲液并从所述第一洗液槽中吸出所述第二缓冲液。Inject a second buffer solution into the second washing solution tank and aspirate the second buffer solution from the first washing solution tank.
  18. 根据权利要求17所述的换液方法在细胞染色和颗粒洗涤中的用途，所述细胞染色包括胞内细胞因子染色或流体表面抗原染色。The use of the fluid exchange method according to claim 17, for cell staining and particle washing, wherein the cell staining includes intracellular cytokine staining or fluid surface antigen staining.
  19. 根据权利要求17所述的换液方法在细胞染色和颗粒洗涤中的用途，所述细胞染色的步骤包括，The use of the fluid exchange method according to claim 17 in cell staining and particle washing, the step of cell staining comprising,
    向所述细胞悬液中加入染色剂，获得所述混合细胞悬液；Adding a stain to the cell suspension to obtain the mixed cell suspension;
    使用所述换液方法对所述混合细胞悬液进行洗涤，获得洗涤后的混合细胞悬液；Washing the mixed cell suspension using the liquid exchange method to obtain a washed mixed cell suspension;
    检测所述洗涤后的混合细胞悬液中的光信号强度；Detecting the intensity of the light signal in the washed mixed cell suspension;
    根据所述光信号强度，分析染色结果。The staining result is analyzed based on the light signal intensity.
  20. 根据权利要求19所述的换液方法在细胞染色和颗粒洗涤中的用途，所述染色剂包括用荧光标记的抗体。Use of a fluid exchange method according to claim 19 in cell staining and particle washing, said staining agent comprising a fluorescently labeled antibody.
PCT/CN2019/107804 2018-09-27 2019-09-25 Solution changing method, solution changing plate, and uses thereof in cell staining and particle washing WO2020063667A1 (en)

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CN201821575639.2U CN208999446U (en) 2018-09-27 2018-09-27 Miniflow liquid changing device
CN201821575639.2 2018-09-27
CN201811127633.3 2018-09-27
CN201811127633.3A CN109270282A (en) 2018-09-27 2018-09-27 Miniflow liquid changing device and change liquid method

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