CN107314965A - The Sample pretreatment method detected based on streaming combination ICP MS single cell proteins - Google Patents
The Sample pretreatment method detected based on streaming combination ICP MS single cell proteins Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
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- G01N15/14—Electro-optical investigation, e.g. flow cytometers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N2015/1486—Counting the particles
Abstract
The invention discloses a kind of Sample pretreatment method detected based on streaming combination ICP MS single cell proteins, belong to the Sample Pretreatment Technique field of flow cytometry.The present invention includes the collection and transport of (1) whole blood sample, (2) PBMC cell separations, (3) cytoactive is dyed, (4) cytositimulation, (5) cell is fixed, (6) surface antibody is dyed, (7) step such as the dyeing of intracellular phosphorylated protein and (8) unicellular mark, the antibody that metallic element label is marked is combined with cell surface antigen, mixed the cell marked with the beads for being used to standardize as internal reference, dead cell and living cells are distinguished in pretreatment process, in case dead cell quantity occur excessively influences the analysis and elaboration of experimental result, distinguish unicellular and cell dimer, tripolymer even polymer, it is well positioned to meet streaming combination ICP MS single cell protein detection demands.
Description
Technical field
The invention belongs to the Sample Pretreatment Technique field of flow cytometry, streaming is based on more specifically to one kind
The pre-treating method of the Sample pretreatment method, more particularly to human peripheral sample that are detected with reference to ICP-MS single cell proteins.
Background technology
Flow cytometry is the cell that a kind of use laser beam excites single file to flow, to its scattered light and the fluorescence carried
Detected, thus complete to carry out the single-row cell in quick straight line flow regime or biologic grain one by one, it is multi-parameter, quick
Qualitative and quantitative analysis or sorting technology, with detection speed is fast, measurement parameter is more, gathered data amount is big, analysis comprehensively,
The features such as separating purity is high, method is flexible, be widely used in clinical medicine, cytology, biology, microbiology, pharmaceutics,
The fields such as genesiology.As one of single cell technology most generally used, streaming technology always in each research field of biology all
Have a wide range of applications.All the time, streaming technology is the important means for analyzing complex cell colony, and it can be realized to cell
Multi parameter analysis is carried out, so as to distinguish different types of cell；It can be detected the number of parameter, also determine us for institute
Study the degree of understanding of population heterogeneity.
The sample-pretreating method of current flow cytometry is substantially carry out fluorochrome label, specific pre-treating method
For：1) 2 × 10^6 cell is collected, is resuspended with PBS cold 1mL, 1500rpm centrifugation 5min abandon supernatant；
2) pat and mix cell mass, be subsequently added the straight target CD45 antibody of 1uL fluorescence, mix, lucifuge is incubated on ice
15min；
3) 1mL PBS are added cell is resuspended, 1500rpm centrifugation 5min abandon supernatant；
4) the neutral paraformaldehyde (notes of 1mL 1% are added：4% paraformaldehyde is diluted into 1% concentration with PBS to use i.e.
Can) cell is resuspended, lucifuge fixes 15min, and subsequent 1500rpm centrifuges 5min, abandons supernatant；
5) 1mL PBS are added cell is resuspended, 1500rpm centrifugation 5min abandon supernatant；
6) cell is resuspended with PBSs of the 1mL containing 0.09% saponin, on ice avoid light place 10min, 1500rpm centrifugation 5min are abandoned
Supernatant；
7) 1mL PBS are added cell is resuspended, 1500rpm centrifugation 5min abandon supernatant；
8) cell is resuspended with 200uLPBS, adds Hsp70 antibody, on ice avoid light place 30min, then it is heavy with PBS cold 1mL
Outstanding cell, 1500rpm centrifugation 5min, abandons supernatant, washes away uncombined primary antibody；
9) 1mL PBS are added cell is resuspended, 1500rpm centrifugation 5min abandon supernatant；
10) cell is resuspended with 200uL PBS, adds the secondary antibody of FITC marks, on ice after avoid light place 40min, then use 1mL
Cell is resuspended in cold PBS, and 1500rpm centrifugation 5min abandon supernatant, wash away uncombined secondary antibody；
11) 1mL PBS are added cell is resuspended, 1500rpm centrifugation 5min abandon supernatant；
12) cell is resuspended with 200uL PBS, lucifuge is standby；Upper machine testing, per 5000-50000 cell of sample count.
The mark of fluorescent dye is different from the principle of metal label mark cell, and processing method is also totally different.Therefore existing mark
The sample that method carries out pre-treatment can not be unicellular applied to human peripheral sample is carried out based on streaming combination ICP-MS well
Protein Detection.The technique study retardance of Sample pretreatment, will have a strong impact on the development and application of novel detection method detection technique.
The content of the invention
1. the problem of solving
It can not be applied to metal label for existing flow cytometry sample-pretreating method and mark asking for cell detection
Topic, the present invention provides a kind of Sample pretreatment method detected based on streaming combination ICP-MS single cell proteins, by metallic element
The antibody of label mark is combined with cell surface antigen, and the cell marked is mixed with being used for the beads of standardization as internal reference
Close, dead cell and living cells distinguished in pretreatment process, so as not to occur dead cell quantity excessively influence the analysis of experimental result with
Illustrate, distinguish unicellular and cell dimer, tripolymer even polymer, be well positioned to meet streaming combination ICP-MS unicellular
Protein Detection demand.
2. technical scheme
In order to solve the above problems, the technical solution adopted in the present invention is as follows：
The Sample pretreatment method detected based on streaming combination ICP-MS single cell proteins, is comprised the following steps：
(1) collection and transport of whole blood sample：Gather and anti-coagulants is added in whole blood sample, sample, normal temperature preserves transport；
(2) PBMC cell separations：The cell in whole blood sample is separated using Ficoll lymphocyte separation mediums；
(3) cytoactive is dyed：The cell of separation is dyed using cis-platinum, dyeing time control is in 5-10min, dye
Supernatant is removed in centrifugation after color terminates, and cell is resuspended；
(4) cytositimulation：Cell is transferred to 15~30min of culture in incubator, cell is recovered " tranquillization " state, and
Packet stimulation is carried out to cell according to appropriate stimulant and stimulating method；
(5) cell is fixed：PFA is carried out to the cell being resuspended in step (3) to fix, freeze；
(6) surface antibody is dyed：After cell PFA is fixed, frozen melts, it is resuspended, adds cell dyeing buffer solution, from
The heart is removed after supernatant, is added Blocking Mix and is resuspended, and normal temperature adds cocktail after standing and mixed；
(7) intracellular phosphorylated protein is dyed：Cell after surface antibody is dyed uses cell with after PBS and methyl alcohol process
Staining Buffer are resuspended, and add phospho-AB cocktail and mix, normal temperature uses Cell Staining after being incubated
Supernatant is removed in Buffer cleanings；
(8) unicellular mark：Cell after being dyed in step (7) is resuspended with PBS, is added dropwise to containing metallic element
The cell intercalation solution of label is marked, and is then carried out respectively with Cell Staining Buffer and water
Cell, is finally resuspended in the water containing EQ beads and preserves by cleaning.
Further, the temperature range that whole blood sample normal temperature is preserved in the step (1) is 15-35 DEG C.
Further, PBMC cell separations are concretely comprised the following steps in step (2)：Will with physiological saline/PBS/Hanks liquid
Whole blood sample 1：After 1 dilution, it is transferred in the centrifuge tube containing 1 times of volume Ficoll lymphocyte separation medium, centrifuges, in sucking-off
The PBMC cells of layer white are transferred in new centrifuge tube, after serum-free DMEM/1640 eccentric cleanings, then with serum-free
Cell precipitation is resuspended in DMEM/1640.
Further, the stimulant described in step (4) can cause cell protein and gene regulation to change to be any
Medicine and kinases, including but not limited to adriamycin, taxol, interferon, growth hormone, the mode of stimulation is outstanding in cell
It is directly added into liquid, it is therefore an objective to stimulate cell to change, and carries out follow-up stimulant and be stimulated cell protein and base
Because of the interactively of change.
Further, in step (5) cell fix concretely comprise the following steps：The cell being resuspended in step (3) is added etc.
2 × fixer of volume is mixed, and ice-cold Cell Staining Buffer are added after incubation, is then centrifuged for separation, with freezing
Cell is resuspended in liquid.
Further, the Blocking Mix solution of step (6) includes Fc Receptor Blocking
Solution and Cell Staning Buffer, both volume ratios are 1:10；Contain antibody in cocktail solution.
Further, what the intracellular phosphorylated protein in step (7) was dyed concretely comprises the following steps：After surface antibody is dyed
Cell mixed with PBS after place on ice, then mixed plus the methanol of precooling, place more than 15min on ice, centrifugation is abandoned
Clearly, it is resuspended with cell staining Buffer, adds isometric phospho-AB cocktail and mix.
Further, the metallic element label in step (8) is Ir or Rh.
3. beneficial effect
Compared to prior art, beneficial effects of the present invention are：
(1) present invention antibody that marks metallic element label is combined with cell surface antigen, by the cell marked and
The beads mixing for being used to standardize as internal reference, dead cell and living cells are distinguished with cis-platinum dyeing, in order to avoid there is dead cell quantity
The analysis and elaboration of excessive influence experimental result；
(2) present invention can be used for preparing the human peripheral blood cell's sample detected based on streaming combination ICP-MS single cell proteins
This, for the detection of individual cell level polyprotein signal, method processing of the invention is convenient, and processing method used, reagent are all
Common method, reagent, it is easy to operate；
(3) present invention is marked using isotope metal marker method, overcomes the glimmering of traditional fluorescent marker method presence
Optical signal is easily quenched, easily bleached, and the weak point such as non-specific background's height；
(4) method that the present invention is provided can mark 30-60 kind metals on a cell, and prior art can only be one
A small amount of metal is marked on individual cell；
(5) present invention can mark cell surface protein, internal albumen, gene, mRNA simultaneously by metal, and existing skill
Art can only mark surface or internal albumen.
Brief description of the drawings
Fig. 1 is the schematic flow sheet of pre-treating method in the present invention；
Embodiment
The present invention is further described below with reference to specific embodiment.
Embodiment 1
As shown in figure 1, based on streaming combination ICP-MS single cell proteins detect Sample pretreatment method the step of include
Herein below：
(1) processing of human peripheral sample (extracts mononuclearcell-cytoactive dyeing-cytositimulation-thin with freezing
Born of the same parents fix)
1. reagent prepares.A. reagent is balanced to normal temperature：Ficoll lymphocyte separation mediums；Ca2+/Mg2+Free PBS；2×
Fixation Solution (3.2%PFA in PBS)；Human red blood cells lysate (optional)；Cell Staining Buffer
(0.5%BSA+0.02%NaN3in PBS).B. the reagent of preheating is needed：FBS Free DMEM/1640(37℃).C. will be with
Lower reagent is placed on ice：Cell Staining Buffer containing 10%DMSO；Cell Staining Buffer；
Cisplatin 5mM；
2. the collection and transport of whole blood sample.Blood specimen collection is the important step of Pre-analysis quality control.Recommend to use
Heparin or citrate are as anti-coagulants, using heparin as anti-coagulants in the present embodiment, according to the standardized amount in hospital
To use；EDTA easily with metal label element chelating affect antibody labeling, so the PBMC samples extracted in EDTA anticoagulated whole bloods
This may need extra washing step.Generally, whole blood sample is needed in 4 hours to be sent to laboratory, normal temperature fortune
It is defeated；(22 degrees Celsius optimal, not below 15 degree or higher than 35 degree)；
3.PBMC cell separations.With physiological saline/PBS/Hanks liquid (mixed liquor of three, generally according to 1:1:1 body
Product after mixing than using) by whole blood sample 1：After 1 dilution, slowly it is added into and is drenched containing 1 times of volume Ficoll with Dispette
In the centrifuge tube of bar cell separation liquid, note tilting centrifuge tube, should not rock, in order to avoid disturbance liquid level influence layering.Normal temperature
400g, brake speed is set to 5, centrifuges 20min；The PBMC cells for suctioning out middle level white are transferred in new 15mL centrifuge tubes.Again
With the serum-free DMEM/1640 of preheating, 300g at room temperature, after 5min eccentric cleanings cell one time, the serum-free preheated with 1mL
Cell precipitation is resuspended in DMEM/1640.It if red blood cell is too many, can be handled with erythrocyte cracked liquid, but larger is influenceed on cytoactive,
If therefore being used with caution when need to be detected to intracellular protein phosphorylation.
4. cytoactive (cis-platinum) is dyed.In the processing procedure of short time (5~10min), cis-platinum cannot be introduced into living cells
Inside, therefore intracellular cis-platinum signal is very weak.And a large amount of albumen and cis-platinum covalent bond in dead cell, stronger signal can be presented,
Cytoactive can be distinguished with this.Cell density is adjusted to 1 × 107/ below mL, is mixed, 37 with the final concentration of 5uM of 1uL cis-platinum
Degree, is incubated 5min；Add the Cell Staining of the normal temperature of 2~5 times of volumes (3 times of volumes are used in the present embodiment)
Buffer, mixes and stops mark reaction, and normal temperature 300g centrifuges 5min, goes after supernatant to use the Cell Staining of normal temperature
Buffer (fixed use) is resuspended or the culture medium (stimulation with) of preheating is resuspended.All Cell used in vital staining step
All contain 0.5%BSA+0.02%NaN in Staining Buffer3。
5. cytositimulation.By cell be transferred in incubator cultivate 20min (typically as needed come select culture when
Between, 15~30min), cell is recovered " tranquillization " state.And cell is carried out according to appropriate stimulant and stimulating method
Packet is stimulated, using gamma interferon as stimulant in the present embodiment, is added in cell solution and is well mixed, and is subsequently used for thin
Born of the same parents fix experiment, and general recommendations is used after cytositimulation in 2 hours.
6. cell is fixed.It is resuspended and mixes cell suspension；Add 2 × fixer (PBS containing 3.2%PFA) of equimultiple volume
Mixed immediately with pipettor piping and druming or vortex oscillation afterwards, to reduce cell formation dimer.It is incubated at room temperature 10min.Add 4mL ice
Cold Cell Staining Buffer fix reaction to slow down PFA.4 DEG C, 600g centrifuges 5min.Remove supernatant.1mL is added to freeze
Cell is resuspended in liquid storage (the Cell Staning Buffer containing 10%DMSO), and being put on ice for standing is used to subsequently dye or put
- 80 degree refrigerators are put into after standing several minutes on ice to freeze.Sample, which should be noted that, avoids multigelation.In pretreatment process, lead to
Cross under the microscope, the method for cell count, analyze how many dimer, tripolymer etc. in every 1000 cells.
(2) antibody of metal label mark is combined (cell surface antibodies dyeing-Intracellular phosphorylation albumen dye with cell
Color-unicellular mark)
1. surface antibody is dyed.For the cell fixed, frozen with PFA, after melting on ice or in cold bath,
Cell is resuspended in Vortex, takes 1mL (106) cell solution to add 2mL Cell Staning Buffer, 600g 5min centrifugations
Supernatant is removed, 50uL Blocking Mix (every part of human PBMC's (PMNC) sample is added：5uL Fc Receptor
Blocking Solution+50uL CSB, take 50uL therein) it is resuspended, room temperature 10min.Often pipe adds 50uL
Cocktail (each antibody consumption of single sample is 1.1uL, cumulative volume 55uL, takes 5uL therein, see the table below) is mixed, often
Temperature 30 minutes.2mL Cell Staining Buffer, normal temperature 500g centrifugation 5min are added, supernatant is removed and is repeated once.
Table 1cocktail constituent
2. intracellular phosphorylated protein is dyed.By above the step of after, add 2mL PBS, 500g centrifugation 5min, go
Clearly.100uL PBS are added, gently Votex mixes cell, placed 3 minutes on ice.The methanol of 1mL precoolings is added, pipette tips are used immediately
Blow even, more than 15min is placed on ice, add 2mL PBS, 800g is centrifuged 5 minutes, abandons supernatant.Add 2mL Cell Staining
Buffer, 800g centrifuge 5min, abandon supernatant.Each sample is resuspended with 50uL cell staining Buffer, adds 50uL phosphorus
Antibody cocktail (the same surface antibody of compound method) is acidified, is mixed, normal temperature is incubated 30 minutes.Add 2mL Cell
Staining Buffer, 800g centrifugation 5min, abandon supernatant.
The unicellular marks of 3.Cell Intercalation.Add 2mL Cell Staining Buffer, normal temperature 500g
5min is centrifuged, supernatant is removed.Add 100ul PBS and cell is resuspended, 1mL cell intercalation are added dropwise in vibration
Solution (adds final concentration of 125nM Ir (iridium) or 500nM Rh (rhodium members in fix and perm buffer
Element), each amount of samples 1mL).2mL Cell Staing Buffe and 2mL water is used respectively, and 800g centrifugation 5min clean cell
After 1 time and 3 times, the water resuspension that 1mL contains 10%EQ beads is added, sample is placed on ice.So far, Sample pretreatment is complete
Into available for subsequent detection.
Streaming combination ICP-MS single cell protein detection method is marked based on metal isotope, its step is：
(1) sample is marked：The specific antibody marked as the method previously described using metallic element, is used as target expression
Reporter；Wherein it is used for the metallic element marked, its atomic mass is usually used in the metal of mark between 88-210Da
Element is Ir (iridium) to be respectively adopted in lanthanide element, the present embodiment or cell is marked Rh (rhodium element).
(2) atomization process：The sample solution after mark is uniformly sent into atomizer with peristaltic pump and carries out atomization process, is gone
Except moisture therein；Atomization temperature is 200 DEG C or so, and the flow channel for liquids in atomizer is ar gas environment, argon gas flow velocity 0.15L/
min。
(3) plasma is ionized：It is unicellular atomization hanging drop enter plasma after gasified, atomization, ionization, formed from
Sub- cloud；Temperature range is 5000K in plasma.
(4) unionization material and photon are removed：Removed using the combination of deflecting electric field and accelerating field in ion cloud stream
The material and photon of the unionization of presence；
(5) ICP-MS is detected：With the atomic mass spectrum of ICP-MS observation individual cells, (ICP-MS detection whole process is in vacuum shape
Carried out under state), the data that atomic mass is composed are converted into cell surface and internal signaling molecule data, and pass through specialty analysis
Software is analyzed the data of acquisition, so as to realize the close-up to cell phenotype and signal network.
Conventional flow cytometer under sheath fluid wrapping, is led to by fluorochrome labeled antibodies, then antibody and antigen binding
Cross flow chamber, form single cell suspension, through laser, and by forward-scattering angle, lateral scattering angle, fluorescence excitation signal information
Computer analysis is passed to via PMT.Originally metallic element label is practiced instead of fluorescent dye label, by nebulizer
Single cell suspension is formed, is ionized single cell suspension by plasma torch, and have TOF to be detected.Due to skill of the present invention
The use of art so that single cell protein white level testing result data volume and data dimension increase sharply, thus during analyze data, Ke Yiying
With a variety of dimension-reduction algorithms, cluster algorithm.
For the analysis of liquid sample, need to remove moisture therein as much as possible before plasma ionizations are entered.
Therefore Nebulizer effect is and is atomized sample, then by being heated to 200-250 DEG C or so of spray chamber, enters
plasma.There is capillary built in one at Nebulizer centers, are sample liquid runner.Microcapillary tube runner is ar gas environment.Argon gas
Flow velocity 0.15-0.35L/min.When sample outflow nebulizer is sophisticated, the shearing force of argon gas stream causes sample flow is formed small
The globule, that is, be atomized.The present invention uses flow type cell principle isolated mononuclear cell, i.e. cell to be atomized into single-cell suspension culture, warp
By argon plasma, breaking of covalent bonds produces free atom, and complete charging process.ICP Torch torch pipes mainly have three-layered node
Structure, outer layer is called outer tube, next to that inner tube, middle is central tube.What is led in outer tube is the argon gas of big flow, is called cooling
Gas, the cooling air lift supply continuous Ar atoms in plasma gas source source, constantly ionization heat release in the plasma, generation
The generating collision in radio-frequency coil of Ar ions, so as to maintain very high temperature, plasma is reserved along with a large amount of ions,
There are many Ar atoms to flow into again, so as to reach a kind of balance.The flow of cooling gas is about 18L/min.Flowed in inner tube
Gas be called auxiliary gas, be also argon gas, its effect is the thrust to plasma flame forward, realize constantly ionization,
Also good central tube, in case too high temperature melts it.It is~1L/min to aid in the flow of gas.Flowed out in central tube
It is the aerosol for the sample solution discharged from fog chamber.
Enter ICP after unicellular atomization globule outflow spray chamber.ICP torch principle is to work as to apply on induction coil
Plus during high-frequency electric field, for some reason (such as electric spark) in plasma working gas partial ionization produce band electrochondria
Son does high-speed motion in the presence of high-frequency alternating electromagnetic field, and collision gas atom is allowed to rapid, a large amount of ionization, forms snowslide
Formula is discharged, and ionized gas forms the vortex of closed annular on the section perpendicular to magnetic direction, is formed in induction coil
Equivalent to transformer secondary coil and with the induction coil coupling equivalent to primary coil, what this high frequency induction current was produced
Gas is heated, ionized again by high temperature, and the plasma flame square of the stabilization in the mouth of pipe one torch-like of formation.Its feature is as follows：
(1) operating temperature it is high, while working gas is inert gas (argon gas), therefore atomization condition is good, is conducive to
Most elements are had very high sensitivity by the decomposition of infusible compound and exciting for element；
(2) due to the presence of Kelvin effect, stability is high, and spontaneous imbibition phenomena is small, and setting-out line scope is wide；
(3) because electron density is high, so interference is smaller caused by the ionization of alkali metal；
(4) ICP belongs to electrodless discharge, in the absence of electrode fouling phenomenon；
(5) ICP flow rate of carrier gas is relatively low, is conducive to sample fully to be excited in centre gangway, and consumption sample amount is also less；
(6) using inert gas (argon gas) as working gas, thus spectral background interference is few
When unicellular hanging drop enters plasma, temperature range is 5000-10000K in plasma.Therefore enter plasma's
Unicellular hanging drop is thus unicellular to be converted into an ion cloud by transient evaporation, atomization, ionization.
Material and photon comprising a large amount of unionizations etc., if not filtering out, are easily attached in ion cloud stream after ionization
Apparatus structure, causes detection signal drift.If wherein photon reaching detector will be mistaken for measured ion to be checked, cause detection
Result precision declines.Therefore, before TOF detections are entered, unionization material therein and photon need to be removed.Utilize deflection electricity
The combination of field and accelerating field is that can be achieved.Sampling spiroid effect is the carrier gas stream from Plasma Center passage, i.e. ion
The most of suction taper hole of stream, into first order vacuum chamber.Sampling spiroid is generally made up of metals such as Ni, Al, Cu, Pt.
Detection technique whole process requires vacuum state.Because with reference to mass-spectrometric technique require ion have longer average freedom
Journey, the probability so as to ion with other ion, molecule, atomic collision when by detector is minimum, and vacuum is directly affected
Ion transmission efficiency, mass spectrum waveform and detector life-span.
The principle screening atomic mass of present invention application tetrode mass analyzer.The effect of tetrode is based at four
Space between electrode produces a special electromagnetic field changed over time, only gives the ion ability of charge-mass ratio (m/z)
Obtain stable path and by pole rod, from other end outgoing, other ions will be deflected too, be collided with pole rod, and in pole
It is neutralized and loses on rod.Removed by tetrode after common bio-element, limit the detection range (88- of the atomic weight of detection
210Da), resolution ratio is high.The larger atom of selective mechanisms atomic mass, filters out the small element of atomic weight, retains wider original
While son amount detection range, because the content of the dvielement in the cell is extremely low, so the background of signal is extremely low.Metallic element
The selection of isotope is not limited to lanthanide series metal.Can be atomic mass between 88-210Da and chelate safety non-toxic
The arbitrary element that can be synthesized.Metal label element species are enriched, and number of channels increases sharply, and information content is also doubled and redoubled.Metal label
Can be by metallo-chelate and antibody binding.
Detected based on flight time (Time of Flight) principle.With inductive couple plasma mass spectrum (ICP-MS)
The atomic mass spectrum of individual cells is observed, the quality of atom is presented with the signaling molecule data of cell surface and inside and corresponded
Relation, that is, detect 1 atomic mass signal and just represent a cell surface and internal signaling molecule, finally by atom
The data of mass spectrum are converted to cell surface and internal signaling molecule data, and pass through data of the specialty analysis software to acquisition
Analyzed, so as to realize the close-up to cell phenotype and signal network.
The increase of signaling molecule sense channel quantity, can be achieved to obtain bigger information content from single sample, is unicellular
The research of proteomics provides new means.But the surge of data volume and data dimension causes the number of original conventional flow
Data result can not be presented well according to analysis chart.Enter line number using a variety of dimension-reduction algorithms, cluster algorithm, clustering algorithm etc.
According to analysis and visualization.A large amount of related algorithm document reports are had at present.As SPADE, DREVI, ACCENSE, XShift,
ViSNE and Phenograp.
Claims (10)
1. the Sample pretreatment method detected based on streaming combination ICP-MS single cell proteins, it is characterised in that：Including following step
Suddenly：
(1) collection and transport of whole blood sample：Gather and anti-coagulants is added in whole blood sample, sample, normal temperature preserves transport；
(2) PBMC cell separations：The cell in whole blood sample is separated using Ficoll lymphocyte separation mediums；
(3) cytoactive is dyed：The cell of separation is dyed using cis-platinum, dyeing time control is in 5-10min, dyeing knot
Supernatant is removed in centrifugation after beam, and cell is resuspended；
(4) cytositimulation：Cell is transferred in incubator 15~30min of culture, cell is recovered " tranquillization " state, and according to
Appropriate stimulant and stimulating method carries out packet stimulation to cell；
(5) cell is fixed：PFA is carried out to the cell being resuspended in step (4) to fix, freeze；
(6) surface antibody is dyed：After cell PFA is fixed, frozen melts, it is resuspended, adds cell dyeing buffer solution, centrifugation is gone
After supernatant, add Blocking Mix and be resuspended, normal temperature adds cocktail after standing and mixed；
(7) intracellular phosphorylated protein is dyed：Cell after surface antibody is dyed uses cell with after PBS and methyl alcohol process
Staining Buffer are resuspended, and add phospho-AB cocktail and mix, normal temperature uses Cell Staining after being incubated
Supernatant is removed in Buffer cleanings；
(8) unicellular mark：Cell after being dyed in step (7) is resuspended with PBS, is added dropwise to containing metallic element mark
The cell intercalation solution of thing is marked, and is then carried out clearly with Cell Staining Buffer and water respectively
Wash, finally cell is resuspended in the water containing EQ beads and preserved.
2. the Sample pretreatment method according to claim 1 detected based on streaming combination ICP-MS single cell proteins, its
It is characterised by：The temperature range that whole blood sample normal temperature is preserved in the step (1) is 15-35 DEG C.
3. the Sample pretreatment method according to claim 1 detected based on streaming combination ICP-MS single cell proteins, its
It is characterised by：Anti-coagulants in the step (1) is heparin or citrate.
4. the Sample pretreatment method according to claim 1 detected based on streaming combination ICP-MS single cell proteins, its
It is characterised by：PBMC cell separations concretely comprises the following steps in step (2)：With physiological saline/PBS/Hanks liquid by whole blood sample 1：
After 1 dilution, it is transferred in the centrifuge tube containing 1 times of volume Ficoll lymphocyte separation medium, centrifuges, suctions out the PBMC of middle level white
Cell is transferred in new centrifuge tube, after serum-free DMEM/1640 eccentric cleanings, then is resuspended with the DMEM/1640 of serum-free
Cell precipitation.
5. before the sample detected based on streaming combination ICP-MS single cell proteins according to any one in claim 1-4
Processing method, it is characterised in that：Stimulant described in step (4) is the medicine that cell protein and gene regulation can be caused to change
Thing and kinases, including but not limited to adriamycin, taxol, interferon, growth hormone.
6. the Sample pretreatment method according to claim 5 detected based on streaming combination ICP-MS single cell proteins, its
It is characterised by：What cell was fixed in step (5) concretely comprises the following steps：By step (3) Gu in the cell that is resuspended add isometric 2 ×
Determine liquid mixing, 0-4 DEG C of Cell Staining Buffer are added after incubation, separation is then centrifuged for, cell is resuspended with frozen stock solution.
7. the Sample pretreatment method according to claim 6 detected based on streaming combination ICP-MS single cell proteins, its
It is characterised by：The Blocking Mix solution of step (6) includes Fc Receptor Blocking Solution and Cell
Staining Buffer, both volume ratios are 1:10；Contain antibody in cocktail solution.
8. the Sample pretreatment method according to claim 7 detected based on streaming combination ICP-MS single cell proteins, its
It is characterised by：Contain 0.5%BSA+0.02%NaN in the vital staining culture medium of step (6)3。
9. the Sample pretreatment method according to claim 6 detected based on streaming combination ICP-MS single cell proteins, its
It is characterised by：What the intracellular phosphorylated protein in step (7) was dyed concretely comprises the following steps：Cell after surface antibody is dyed is used
PBS is placed on ice after mixing, and is then mixed plus the methanol of precooling, places more than 15min on ice, and centrifugation abandons supernatant, uses cell
Staining Buffer are resuspended, and add isometric phospho-AB cocktail and mix.
10. the Sample pretreatment method according to claim 6 detected based on streaming combination ICP-MS single cell proteins, its
It is characterised by：Metallic element label in step (8) is Ir or Rh.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108508196A (en) * | 2018-04-13 | 2018-09-07 | 沈阳汇敏源生物科技有限责任公司 | A kind of kit and detection method of detection people's regulatory T cells hypotype |
| CN109323908A (en) * | 2018-10-10 | 2019-02-12 | 浙江大学 | A kind of detection method for mass spectrum streaming technology |
| CN109997842A (en) * | 2019-04-10 | 2019-07-12 | 天津中科生物科技有限公司 | Cell-preservation liquid and preparation method thereof and its application in streaming and antibody test |
| CN112834411A (en) * | 2021-01-07 | 2021-05-25 | 上海交通大学 | Metal nanoprobe applied to mass flow technology, preparation method and application |
| CN113033389A (en) * | 2021-03-23 | 2021-06-25 | 天津凌视科技有限公司 | Method and system for image recognition by using high-speed imaging device |
| CN114441417A (en) * | 2022-01-18 | 2022-05-06 | 重庆生命知源科技有限公司 | Flow cytometer based detection method for trace cell phosphorylation protein expression |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH05290795A (en) * | 1992-04-13 | 1993-11-05 | Seiko Instr Inc | Inductive coupling plasma mass spectrometer |
| CN103105326A (en) * | 2012-12-30 | 2013-05-15 | 上海市内分泌代谢病研究所 | Method for surface staining and intracellular staining of Th cells and application of method |
| US20140070092A1 (en) * | 2001-07-17 | 2014-03-13 | Perkinelmer Health Sciences, Inc. | Elemental flow cytometer |
| CN102876631B (en) * | 2012-10-09 | 2014-04-09 | 博雅干细胞科技有限公司 | Method for separating immune cells from blood and application of method to disease treatment |
| CN105683397A (en) * | 2013-09-04 | 2016-06-15 | 富鲁达公司 | Proximity assays for detecting nucleic acids and proteins in a single cell |
-
2017
- 2017-06-22 CN CN201710479767.0A patent/CN107314965B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH05290795A (en) * | 1992-04-13 | 1993-11-05 | Seiko Instr Inc | Inductive coupling plasma mass spectrometer |
| US20140070092A1 (en) * | 2001-07-17 | 2014-03-13 | Perkinelmer Health Sciences, Inc. | Elemental flow cytometer |
| CN102876631B (en) * | 2012-10-09 | 2014-04-09 | 博雅干细胞科技有限公司 | Method for separating immune cells from blood and application of method to disease treatment |
| CN103105326A (en) * | 2012-12-30 | 2013-05-15 | 上海市内分泌代谢病研究所 | Method for surface staining and intracellular staining of Th cells and application of method |
| CN105683397A (en) * | 2013-09-04 | 2016-06-15 | 富鲁达公司 | Proximity assays for detecting nucleic acids and proteins in a single cell |
Non-Patent Citations (2)
| Title |
|---|
| CHARLOTTE GIESEN ET AL.: ""History of inductively coupled plasma mass spectrometry-based immunoassays"", 《SPECTROCHIMICA ACTA PART B》 * |
| OLGA ORNATSKY ET AL.: ""Highly multiparametric analysis by mass cytometry"", 《JOURNAL OF IMMUNOLOGICAL METHODS》 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108508196A (en) * | 2018-04-13 | 2018-09-07 | 沈阳汇敏源生物科技有限责任公司 | A kind of kit and detection method of detection people's regulatory T cells hypotype |
| CN108508196B (en) * | 2018-04-13 | 2020-10-27 | 何韶衡 | Kit and method for detecting human regulatory T cell subtype |
| CN109323908A (en) * | 2018-10-10 | 2019-02-12 | 浙江大学 | A kind of detection method for mass spectrum streaming technology |
| CN109997842A (en) * | 2019-04-10 | 2019-07-12 | 天津中科生物科技有限公司 | Cell-preservation liquid and preparation method thereof and its application in streaming and antibody test |
| CN109997842B (en) * | 2019-04-10 | 2021-08-13 | 天津中科生物科技有限公司 | Cell preservation solution, preparation method thereof and application thereof in flow-type and antibody test |
| CN112834411A (en) * | 2021-01-07 | 2021-05-25 | 上海交通大学 | Metal nanoprobe applied to mass flow technology, preparation method and application |
| CN112834411B (en) * | 2021-01-07 | 2022-05-27 | 上海交通大学 | Metal nanoprobe applied to mass flow technology, preparation method and application |
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| CN113033389B (en) * | 2021-03-23 | 2022-12-16 | 天津凌视科技有限公司 | Method and system for image recognition by using high-speed imaging device |
| CN114441417A (en) * | 2022-01-18 | 2022-05-06 | 重庆生命知源科技有限公司 | Flow cytometer based detection method for trace cell phosphorylation protein expression |
| CN114441417B (en) * | 2022-01-18 | 2023-02-07 | 重庆生命知源科技有限公司 | Flow cytometer based detection method for trace cell phosphorylation protein expression |
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