CN102288531B - Method and device for filtering, dyeing and counting cells - Google Patents
Method and device for filtering, dyeing and counting cells Download PDFInfo
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- CN102288531B CN102288531B CN 201110100618 CN201110100618A CN102288531B CN 102288531 B CN102288531 B CN 102288531B CN 201110100618 CN201110100618 CN 201110100618 CN 201110100618 A CN201110100618 A CN 201110100618A CN 102288531 B CN102288531 B CN 102288531B
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Abstract
The invention discloses a method and a device for filtering, dyeing and counting cells. The method comprises the following steps: preparing isometric cells needing to be counted in an experimental group and at least four groups of cell suspension liquids with known quantities in a standard group, respectively filtering the cells, cleaning the cells by using a phosphate buffering liquid, further, soaking the cells by adopting a crystal violet solution, absorbing the crystal violet solution, then cleaning the cells by using the phosphate buffering liquid, flushing the cells by using an acetic acid solution, and then collecting a filtrate, measuring optical density at a wavelength of 570 nm; combining the known quantities of the cells in the standard group with the optical density to draw a standard curve, and finally, calculating the quantities of the cells in the experimental group according to the standard curve and the optical density of cell samples in the experimental group. In the invention, the beneficial effects that the influence of a subjective degree factor to a result is reduced, and the automatic batch processing is easily achieved, so as to improve measurement precision and result parallelism are shown.
Description
Technical field
The present invention relates to a kind of measuring method and device, specifically, is about Cytometric method and device.
Background technology
Cell count is at life science, the technology of especially in cellular incubation related experiment and production, often using.Existing method for cell count respectively has limitation: classical blood counting chamber method and series derivatives method thereof (be like application number 01213605.0 the said method for cell count of Chinese patent) are though measure directly perceived; But, cell can produce counting error owing to disperseing reasons such as the skill level unfavorable or testing staff; And single sample counting is consuming time longer; Can't operate great amount of samples simultaneously, reduce the collimation of experimental result; Though decoration method can satisfy the requirement of batch operation; But often be only applicable to the cell (like Jap.P. WO2006003696) of adherent growth; Cell for suspension growth also needs some operations to accomplish, thereby has influenced the batch processing degree of counting, has reduced the accuracy of counting.
Summary of the invention
In order to overcome the shortcoming that the accuracy of prior art counting is not high, be not easy to realize robotization, the invention provides a kind of simple to operately, subjective number of degrees factor is few, easily is automated the method for cell count of batch processing.
The technical scheme steps that the present invention adopted may further comprise the steps:
1. be ready to need experimental group cell and the standard group cell suspension of at least 4 group dose known amounts of the unknown number of counting; Said cell suspension can directly be used by the suspension growth cell; Or the formation of attached cell process digestion step, each volume of organizing cell suspension is identical;
2. the corresponding filtration unit of each cell suspension sample passes through filtration unit with each group cell suspension respectively, makes cell retention on filter membrane;
3. use 2 times of phosphate buffers of cell suspension (PBS) to clean respectively and be trapped in the cell on the filtration unit with upper volume;
4. add 1 times of concentration of cell suspension respectively and be 0.1%～1% crystal violet solution and in filtration unit, soaked cell 10 minutes～1 hour with upper volume;
5. inhale the purple solution that decrystallizes respectively, with 5 times of cell suspensions with the cell in the upper volume PBS cleaning and filtrating equipment;
6. discharge remaining liquid in the filtration unit respectively;
7. using 1 times of concentration with upper volume of cell suspension respectively is 3%～30% acetum washing and filtering device, collects filtrating;
Respectively with gained filtrating after 3 times of dilutions are more than the volume, use visible spectrophotometer, measurement 570nm wavelength optical density;
9. known cell quantity of combined standard group and optical density drawing standard curve obtain optical density I
StandardWith cell quantity N
StandardRelation formula: I
Standard=a * N
Standard+ b, wherein a is the monobasic linearly dependent coefficient, b is an intercept, all calculates through typical curve and learns;
10. calculate experimental group cell quantity, i.e. N according to the optical density of typical curve and experimental group cell sample
Experiment=(I
Experiment-b)/a.
Described crystal violet solution adopts water, physiological saline or PBS damping fluid to prepare as solvent.
The present invention also provides a kind of device that is used to filter dyeing counting cell method, comprises lye container, dyeing liquor container, destainer container, waste fluid container, filtration unit, pumping system, colorimetric device, T-valve and computing machine.Experimental group and standard group cell suspension are connected with lye container, dyeing liquor container respectively through T-valve; Be connected to filtration unit with the destainer container through T-valve afterwards; The delivery outlet of filtration unit links to each other with colorimetric device through pumping system, and colorimetric device feeds back measurement data in computing machine.After the completion to be detected, waste liquid is rejected in the waste fluid container by the conduit that links to each other with colorimetric device.In above-mentioned each link, by each T-valve of computer control, pumping system and colorimetric device work.
The invention has the beneficial effects as follows: owing to adopt filtration unit pair cell suspension to filter; Entrapped cell; Filter membrane in the filtration unit is as the solid phase supporting carrier; To cell on it dye, wash, processing such as decolouring, finally utilize the optical density of crystal violet eluent to calculate cell quantity.Compared with prior art, the present invention has embodied and has reduced the influence of subjective number of degrees factor to the result, easily is automated batch processing, improve to measure the precision and the beneficial effect of collimation as a result.
Below in conjunction with accompanying drawing and embodiment the present invention is further specified.
Description of drawings
Fig. 1 is a method flow diagram of the present invention;
Fig. 2 is a device synoptic diagram of the present invention;
Among the figure, 1-experimental group and standard group cell suspension, 2-lye container, 3-dyeing liquor container, 4-destainer container, 5-filtration unit, 6-pumping system, 7-colorimetric device, 8-waste fluid container, 9-computing machine, 10-T-valve b, 11-T-valve a, 12-T-valve c.
Embodiment
Method embodiment 1: use the method for the invention to carry out human leukemia cell line K562 cell count
1, prepares human leukemia cell line K562 with every milliliter 8 * 10 of unknown density (experimental group) and known density (standard group)
5, 4 * 10
5, 2 * 10
5, 1 * 10
5, 0.5 * 10
5, 0.25 * 10
5Individual cell;
2, with 0.2 milliliter respectively organize the sample mixing after inject filtration unit 5 respectively, make cell retention on filtration unit 5;
3, open T-valve a11, T-valve b10 and T-valve c12, inject the cell on phosphate buffer (PBS) cleaning and filtrating equipment 5 of 1 milliliter of lye container 2 respectively;
4, opening T-valve a11, T-valve b10 and T-valve c12, to add concentration in 0.5 milliliter of dyeing liquor container 3 be that 0.1% crystal violet solution soaked cell 1 hour in filtration unit;
5, use pumping system 6 to inhale the purple solution that decrystallizes, open T-valve a11, T-valve b10 and T-valve c12 also with the cell in the PBS cleaning and filtrating equipment 5 in 10 milliliters of lye containers 2;
6, discharge remaining liquid in the filtration unit 5 through pumping system 6;
7, open T-valve c12, the cell with on the acetum immersion filtration unit 5 of 10% in 0.5 milliliter of destainer device 4 makes it decolouring, collects destainer;
8, gained is filtrated after 3 times of dilutions, use colorimetric device 7, measure 570nm wavelength optical density, and import data into computing machine 9 like visible spectrophotometer;
9, known cell quantity of combined standard group and optical density drawing standard curve obtain optical density I
StandardWith cell quantity N
StandardRelation formula: I
Standard=a * N
Standard+ b, wherein a is the monobasic linearly dependent coefficient, b is an intercept, all calculates through typical curve and learns;
10, calculate experimental group cell quantity, i.e. N according to the optical density of typical curve and experimental group cell sample
Experiment=(I
Real Test-b)/a.
11, known cell quantity of combined standard group and optical density drawing standard curve obtain optical density I
StandardWith cell quantity N
StandardRelation formula: I
Standard=a * N
Standard+ b, wherein a is the monobasic linearly dependent coefficient, b is an intercept, all calculates through typical curve and learns;
12, calculate experimental group cell quantity, i.e. N according to the optical density of typical curve and experimental group cell sample
Experiment=(I
Real Test-b)/a.
Method embodiment 2: using the method for the invention to carry out people's bone like cell is the MLO-Y4 cell count
1. prepare people's bone like cell and be MLO-Y4 with every milliliter 4 * 10 of unknown density (experimental group) and known density (standard group)
5, 2 * 10
5, 1 * 10
5, 0.5 * 10
5, 0.25 * 10
5Individual cell;
With 0.3 milliliter respectively organize the sample mixing after inject filtration unit 5 respectively, make cell retention on filtration unit 5;
3. open T-valve a11, T-valve b10 and T-valve c12, inject the cell on phosphate buffer (PBS) cleaning and filtrating equipment 5 of 1.5 milliliters of lye containers 2 respectively;
4. opening T-valve a11, T-valve b10 and T-valve c12, to add concentration in 1 milliliter of dyeing liquor container 3 be that 0.1% crystal violet solution soaked cell 1 hour in filtration unit;
5. use pumping system 6 to inhale the purple solution that decrystallizes, open T-valve a11, T-valve b10 and T-valve c12 also with the cell in the PBS cleaning and filtrating equipment 5 in 12 milliliters of lye containers 2;
6. discharge remaining liquid in the filtration unit 5 through pumping system 6;
7. open T-valve c12, the cell with on the acetum immersion filtration unit 5 of 10% in 1 milliliter of destainer device 4 makes it decolouring, collects destainer;
8. gained is filtrated after 5 times of dilutions, use colorimetric device 7, measure 570nm wavelength optical density, and import data into computing machine 9 like visible spectrophotometer;
9. known cell quantity of combined standard group and optical density drawing standard curve obtain optical density I
StandardWith cell quantity N
StandardRelation formula: I
Standard=a * N
Standard+ b, wherein a is the monobasic linearly dependent coefficient, b is an intercept, all calculates through typical curve and learns;
10. calculate experimental group cell quantity, i.e. N according to the optical density of typical curve and experimental group cell sample
Experiment=(I
Experiment-b)/a.
11. known cell quantity of combined standard group and optical density drawing standard curve obtain optical density I
StandardWith cell quantity N
StandardRelation formula: I
Standard=a * N
Standard+ b, wherein a is the monobasic linearly dependent coefficient, b is an intercept, all calculates through typical curve and learns;
12. the optical density according to typical curve and experimental group cell sample is calculated experimental group cell quantity, i.e. N
Experiment=(I
Experiment-b)/a.
Method embodiment 3: using the method for the invention to carry out people's human osteosarcoma cell is the MG-63 cell count
1, to prepare people's human osteosarcoma cell be MG-63 with every milliliter 8 * 10 of unknown density (experimental group) and known density (standard group)
5, 4 * 10
5, 2 * 10
5, 1 * 10
5, 0.5 * 10
5Individual cell;
2, with 0.5 milliliter respectively organize the sample mixing after inject filtration unit 5 respectively, make cell retention on filtration unit 5;
3, open T-valve a11, T-valve b10 and T-valve c12, inject the cell on phosphate buffer (PBS) cleaning and filtrating equipment 5 of 2 milliliters of lye containers 2 respectively;
4, opening T-valve a11, T-valve b10 and T-valve c12, to add concentration in 1.5 milliliters of dyeing liquor containers 3 be that 0.1% crystal violet solution soaked cell 1 hour in filtration unit;
5, use pumping system 6 to inhale the purple solution that decrystallizes, open T-valve a11, T-valve b10 and T-valve c12 also with the cell in the PBS cleaning and filtrating equipment 5 in 15 milliliters of lye containers 2;
6, discharge remaining liquid in the filtration unit 5 through pumping system 6;
7, open T-valve c12, the cell with on the acetum immersion filtration unit 5 of 10% in 1.5 milliliters of destainer devices 4 makes it decolouring, collects destainer;
8, gained is filtrated after 2 times of dilutions, use colorimetric device 7, measure 570nm wavelength optical density, and import data into computing machine 9 like visible spectrophotometer;
9, known cell quantity of combined standard group and optical density drawing standard curve obtain optical density I
StandardWith cell quantity N
StandardRelation formula: I
Standard=a * N
Standard+ b, wherein a is the monobasic linearly dependent coefficient, b is an intercept, all calculates through typical curve and learns;
10, calculate experimental group cell quantity, i.e. N according to the optical density of typical curve and experimental group cell sample
Experiment=(I
Real Test-b)/a.
11, known cell quantity of combined standard group and optical density drawing standard curve obtain optical density I
StandardWith cell quantity N
StandardRelation formula: I
Standard=a * N
Standard+ b, wherein a is the monobasic linearly dependent coefficient, b is an intercept, all calculates through typical curve and learns;
12, calculate experimental group cell quantity, i.e. N according to the optical density of typical curve and experimental group cell sample
Experiment=(I
Real Test-b)/a.
Adopt method pair cell of the present invention to count, in the range of linearity wide (0.25 * 10
5～8 * 10
5), the data result collimation is good, and the 570nm place optical density according to gained equation with one unknown quantity and measurement sample can calculate the cell number in the sample fast and accurately.
Device embodiment:
The present invention is designed for the device that filters dyeing counting cell method simultaneously.Device comprises: lye container, dyeing liquor container, destainer container, waste fluid container, filtration unit, pumping system, colorimetric device, T-valve, computing machine.
Specify as follows:
Experimental group and standard group cell suspension are connected with the container that is used for cell washing, dyeing respectively through T-valve b, and this process is through the realization that is connected of T-valve b and T-valve a (container that is connected cleansing solution and dyeing liquor); Simultaneously, T-valve b is connected with the container of the cell decolouring afterwards that is used to dye, and this process realizes with the connection of T-valve c (container that is connected destainer) through T-valve b.In addition, T-valve c connects filtration unit, and filtration unit is connected with pumping system; Pumping system links to each other with colorimetric device simultaneously, and colorimetric device feeds back measurement data in computing machine.After the completion to be detected, waste liquid is rejected in the container that holds waste liquid by the conduit that links to each other with colorimetric device.In above-mentioned each link, whether by the startup of computer control T-valve a, T-valve b, T-valve c, pumping system and colorimetric system.
Claims (4)
1. a method of filtering the dyeing counting cell is characterized in that comprising the steps:
1) be ready to need experimental group cell and the standard group cell suspension of at least 4 group dose known amounts of the unknown number of counting, each volume of organizing cell suspension is identical;
2) corresponding filtration unit of each cell suspension sample passes through filtration unit with each group cell suspension respectively, makes cell retention on filter membrane;
3) use 2 times of phosphate buffers of cell suspension to clean respectively and be trapped in the cell on the filtration unit with upper volume;
4) adding 1 times of concentration with upper volume of cell suspension respectively is that 0.1%～1% crystal violet solution soaked cell 10 minutes～1 hour in filtration unit;
5) inhale the purple solution that decrystallizes respectively, with 5 times of cell suspensions with the cell in the upper volume phosphate buffer cleaning and filtrating equipment;
6) discharge remaining liquid in the filtration unit respectively;
7) using 1 times of concentration with upper volume of cell suspension respectively is 3%～30% acetum washing and filtering device, collects filtrating;
8) respectively with gained filtrating after 3 times of dilutions are more than the volume, use visible spectrophotometer, measurement 570nm wavelength optical density;
9) known cell quantity of combined standard group and optical density drawing standard curve obtain optical density I
StandardWith cell quantity N
StandardRelation formula: I
Standard=a * N
Standard+ b, wherein a is the monobasic linearly dependent coefficient, b is an intercept, all calculates through typical curve and learns;
10) calculate experimental group cell quantity, i.e. N according to the optical density of typical curve and experimental group cell sample
Experiment=(I
Experiment-b)/a.
2. the method for filtration dyeing counting cell according to claim 1 is characterized in that: described cell suspension is directly used by the suspension growth cell, or attached cell forms through digestion step.
3. the method for filtration dyeing counting cell according to claim 1 is characterized in that: described crystal violet solution adopts water, physiological saline or phosphate buffer to prepare as solvent.
4. filtration dyeing counting cell device of realizing the said method of claim 1; Comprise lye container, dyeing liquor container, destainer container, waste fluid container, filtration unit, pumping system, colorimetric device, T-valve and computing machine; It is characterized in that: experimental group and standard group cell suspension are connected with lye container, dyeing liquor container respectively through first T-valve; Be connected to filtration unit with the destainer container through second T-valve afterwards; The delivery outlet of filtration unit links to each other with colorimetric device through pumping system, and colorimetric device feeds back measurement data in computing machine; After the completion to be detected, waste liquid is rejected in the waste fluid container by the conduit that links to each other with colorimetric device; In above-mentioned each link, by each T-valve of computer control, pumping system and colorimetric device work.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110100618 CN102288531B (en) | 2011-04-21 | 2011-04-21 | Method and device for filtering, dyeing and counting cells |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110100618 CN102288531B (en) | 2011-04-21 | 2011-04-21 | Method and device for filtering, dyeing and counting cells |
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| CN102288531B true CN102288531B (en) | 2012-12-19 |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1294676A (en) * | 1998-02-20 | 2001-05-09 | Xy公司 | Viberatory system for sorting flow cytometer |
| CN1502068A (en) * | 2000-08-02 | 2004-06-02 | ����Τ�����ʹ�˾ | Portable scattering and fluorescence cytometer |
| CN101052867A (en) * | 2004-09-01 | 2007-10-10 | 霍尼韦尔国际公司 | Frequency-multiplexed detection of multiple wavelength light for flow cytometry |
| CN101498645A (en) * | 2009-03-05 | 2009-08-05 | 北京理工大学 | Method for single-cell continuous flow capillary electrophoresis dual-wavelength cell activity detection |
| CN202075202U (en) * | 2011-04-21 | 2011-12-14 | 西北工业大学 | Device for counting cells by filtering and staining |
-
2011
- 2011-04-21 CN CN 201110100618 patent/CN102288531B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1294676A (en) * | 1998-02-20 | 2001-05-09 | Xy公司 | Viberatory system for sorting flow cytometer |
| CN1502068A (en) * | 2000-08-02 | 2004-06-02 | ����Τ�����ʹ�˾ | Portable scattering and fluorescence cytometer |
| CN101052867A (en) * | 2004-09-01 | 2007-10-10 | 霍尼韦尔国际公司 | Frequency-multiplexed detection of multiple wavelength light for flow cytometry |
| CN101498645A (en) * | 2009-03-05 | 2009-08-05 | 北京理工大学 | Method for single-cell continuous flow capillary electrophoresis dual-wavelength cell activity detection |
| CN202075202U (en) * | 2011-04-21 | 2011-12-14 | 西北工业大学 | Device for counting cells by filtering and staining |
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Effective date of registration: 20140814 Address after: 226600, No. 8 Xin Gang Road, Haian Development Zone, Haian County, Jiangsu, Nantong Patentee after: Jiangsu Huaan Scientific Research Devices Co., Ltd. Patentee after: Northwestern Polytechnical University Address before: 710072 Xi'an friendship West Road, Shaanxi, No. 127 Patentee before: Northwestern Polytechnical University |