CN115078019A - High-accuracy cell cycle detection method - Google Patents
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- 238000001514 detection method Methods 0.000 title claims abstract description 39
- 230000022131 cell cycle Effects 0.000 title claims abstract description 26
- 210000004027 cell Anatomy 0.000 claims abstract description 88
- 239000000243 solution Substances 0.000 claims abstract description 49
- 210000002901 mesenchymal stem cell Anatomy 0.000 claims abstract description 29
- 238000000034 method Methods 0.000 claims abstract description 21
- 108090000790 Enzymes Proteins 0.000 claims abstract description 15
- 102000004190 Enzymes Human genes 0.000 claims abstract description 15
- 238000011081 inoculation Methods 0.000 claims abstract description 14
- 239000007788 liquid Substances 0.000 claims abstract description 14
- 238000000684 flow cytometry Methods 0.000 claims abstract description 13
- 238000005406 washing Methods 0.000 claims abstract description 12
- 239000006285 cell suspension Substances 0.000 claims abstract description 11
- 238000004043 dyeing Methods 0.000 claims abstract description 11
- 230000002255 enzymatic effect Effects 0.000 claims abstract description 10
- 239000012192 staining solution Substances 0.000 claims abstract description 10
- 239000006228 supernatant Substances 0.000 claims abstract description 8
- 238000010257 thawing Methods 0.000 claims abstract description 8
- 108010009736 Protein Hydrolysates Proteins 0.000 claims abstract description 5
- 230000008014 freezing Effects 0.000 claims abstract description 5
- 238000007710 freezing Methods 0.000 claims abstract description 5
- 239000000413 hydrolysate Substances 0.000 claims abstract description 5
- 239000000463 material Substances 0.000 claims abstract description 5
- 238000002156 mixing Methods 0.000 claims abstract description 5
- 230000001376 precipitating effect Effects 0.000 claims abstract description 3
- 239000000872 buffer Substances 0.000 claims description 10
- 210000003954 umbilical cord Anatomy 0.000 claims description 7
- 239000011550 stock solution Substances 0.000 claims description 4
- 210000003074 dental pulp Anatomy 0.000 claims description 2
- 239000000834 fixative Substances 0.000 claims description 2
- 210000002826 placenta Anatomy 0.000 claims description 2
- 239000000126 substance Substances 0.000 abstract description 2
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 10
- 239000007853 buffer solution Substances 0.000 description 9
- 238000012360 testing method Methods 0.000 description 9
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 102000006382 Ribonucleases Human genes 0.000 description 4
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- 238000004140 cleaning Methods 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 208000035199 Tetraploidy Diseases 0.000 description 2
- 230000032823 cell division Effects 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000016507 interphase Effects 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000002844 melting Methods 0.000 description 2
- 230000008018 melting Effects 0.000 description 2
- 230000006820 DNA synthesis Effects 0.000 description 1
- 230000035519 G0 Phase Effects 0.000 description 1
- 230000010337 G2 phase Effects 0.000 description 1
- 230000004668 G2/M phase Effects 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 210000004504 adult stem cell Anatomy 0.000 description 1
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- 238000006243 chemical reaction Methods 0.000 description 1
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- 238000011160 research Methods 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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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—Optical investigation techniques, e.g. flow cytometry
- G01N15/1434—Optical arrangements
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/302—Stain compositions
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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
- G01N2015/1006—Investigating individual particles for cytology
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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—Optical investigation techniques, e.g. flow cytometry
- G01N15/1434—Optical arrangements
- G01N2015/144—Imaging characterised by its optical setup
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Abstract
The invention relates to the field of chemical detection, wherein the IPC classification number is G01N33/53, and particularly relates to a high-accuracy cell cycle detection method. The high-accuracy cell cycle detection method comprises the following steps: (1) cell thawing: taking a freezing tube filled with the frozen material for thawing, centrifuging and resuspending to obtain resuscitated cells; (2) cell inoculation: inoculating the revived cells into a culture bottle to obtain inoculated cells; (3) cell fixation: collecting inoculated cells, washing, centrifuging, removing supernatant, precipitating and resuspending, and fixing the cells by using a cell fixing solution; (4) enzymolysis: preparing the fixed cells into cell suspension, and then adding an enzyme solution into the cell suspension to obtain enzymatic hydrolysate; (5) dyeing: adding a staining solution into the enzymatic hydrolysate, mixing uniformly, and incubating in a dark place to obtain a detection solution; (6) and (3) computer detection: and detecting and analyzing the detection liquid by using a flow cytometry analyzer. The method can accurately detect the cell cycle of the mesenchymal stem cells.
Description
Technical Field
The invention relates to the field of chemical detection, wherein the IPC classification number is G01N33/53, and particularly relates to a high-accuracy cell cycle detection method.
Background
Researchers have studied cells a long time ago, and the cell cycle refers to the process from the beginning of growth of new cells produced by cell division to the end of the next cell division to form daughter cells, and is divided into an interphase and a division phase (M phase). The interphase is divided into G1 phase (Gap 1phase), S phase (DNA synthsis phase) and G2 phase (Gap 2phase), wherein G1 phase is used for preparing S phase and mainly synthesizes RNA and ribosome; s phase is mainly DNA and histone; stage G2 is for stage M, and is the late stage of DNA synthesis; the M phase is a continuously variable process in which one mother cell divides into two daughter cells.
The mesenchymal stem cells belong to non-hematopoietic adult stem cells, have wide tissue distribution and self-renewal and differentiation capacity. Patent CN1168984C provides a two-parameter cell cycle analysis method, which can better distinguish the cell cycle, but the fixation effect is not good, and the mesenchymal stem cell cycle cannot be well detected.
Disclosure of Invention
A high-accuracy cell cycle detection method comprises the following steps:
(1) cell thawing: taking the freezing tube filled with the frozen material for thawing, centrifuging and resuspending to obtain resuscitated cells;
(2) cell inoculation: inoculating the revived cells into a culture bottle to obtain inoculated cells;
(3) cell fixation: collecting inoculated cells, washing, centrifuging, removing supernatant, precipitating and resuspending, and fixing the cells by using a cell fixing solution;
(4) enzymolysis: preparing the fixed cells into cell suspension, and then adding an enzyme solution into the cell suspension to obtain enzymatic hydrolysate;
(5) dyeing: adding a staining solution into the enzymatic hydrolysate, mixing uniformly, and incubating in a dark place to obtain a detection solution;
(6) and (3) computer detection: and detecting and analyzing the detection liquid by using a flow cytometry analyzer.
The frozen stock in the step (1) is frozen stock solution and mesenchymal stem cells.
The mesenchymal stem cell is a Pi-generation mesenchymal stem cell, and i is an integer of 1 or more, for example (i ═ 1, 2, 3, 4, 5, 6, and the like).
The mesenchymal stem cells are selected from at least one of human umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, placenta mesenchymal stem cells and dental pulp mesenchymal stem cells.
The melting conditions in the step (1) are as follows: melting in water bath at 37 deg.C for 2 min.
The resuspension conditions in step (1) were: MM3 was resuspended in complete medium, the volume of MM3 complete medium was 5 times the volume of the frozen stock in the frozen tubes.
The fusion degree of the inoculated cells in the step (2) is 75-85%; preferably, the degree of confluency of the cells seeded in step (2) is 80%.
The inoculation density of the resuscitation cell inoculation in the step (2) is 5000- 2 (ii) a Preferably, the inoculation density of the revival cell inoculation in the step (2) is 6000/cm 2 。
The washing solution used in the washing in step (3) is PBS buffer solution.
The PBS buffer was purchased from HyClone under the cat # SH 30256.01.
The washing times in the step (3) are 1-4 times; preferably, the number of washing in step (2) is 2.
Resuspending in step (3) using PBS buffer; in step (3), PBS buffer used for resuspension: inoculating cells: fixed solution 100 μ l: 2.5X 10 6 -7×10 6 The method comprises the following steps: 1 mL; preferably, in step (3), the PBS buffer used for resuspension: inoculating cells: fixative 100 μ l: 4X 10 6 The method comprises the following steps: 1 mL.
The volume concentration of the fixing liquid is 60-80%; preferably, the volume concentration of the fixing solution is 70 percent
The fixing solution is an ethanol water solution precooled at the temperature of-20 ℃, wherein the ethanol water solution is prepared from ethanol and ultrapure water.
The conditions for fixing the cells in the step (3) are as follows: the reaction was refrigerated overnight at 4 ℃.
The preparation method of the cell suspension in step (4) is a method known in the art, i.e., the fixed cells are centrifuged, washed, supernatant removed, and the pellet is resuspended in PBS buffer, wherein the amount of PBS buffer used is the same as the amount of PBS buffer used in the resuspension in step (3).
The enzyme solution: seeded cells in step (2) ═ 3 μ l: 2.5X 10 6 -7×10 6 A plurality of; preferably, the enzyme solution: seeded cells in step (2) ═ 3 μ l: 4X 10 6 And (4) respectively.
The enzyme solution is RNase enzyme solution.
The dyeing liquid is 20-30ug/mL PI dyeing liquid; preferably, the dyeing solution is 25ug/mL of PI dyeing solution.
The volume ratio of the enzyme solution to the dyeing solution is (1-2): 100, respectively; preferably, the volume ratio of the enzyme solution to the staining solution is 1.5: 100.
in the step (5), the temperature is controlled to be 20-26 ℃ away from light, and the time is controlled to be 20-40min away from light; preferably, the light-shielding temperature in the step (5) is 23 ℃ and the light-shielding time is 30 min.
Propidium Iodide (PI) can be combined with intracellular DNA and RNA, and after RNA is digested by RNase enzyme solution, the fluorescence intensity of PI combined with DNA detected by flow cytometry directly reflects the content of the intracellular DNA. Because of the different DNA content in each phase of the cell cycle, normal cells usually have the DNA content of diploid cells (2N) in the G1/G0 phase, and tetraploid cells (4N) in the G2/M phase, and the DNA content of S phase is between that of diploid and tetraploid cells. Therefore, when the DNA content in the cells is detected by the PI staining method of flow cytometry, the proportion of each cell subgroup in different cell cycles can be known. The applicant finds that the volume ratio of the enzyme solution to the dyeing solution is controlled to be (1-2): 100, can better realize the digestion of RNA and the accurate detection of cell cycle, and leads the accuracy of the detection result to be higher.
The method comprises the following steps of computer detection and analysis of the flow cytometry analyzer:
s1, opening the flow cytometry, newly building an experimental method, and cleaning and preheating the instrument;
s2 setting parameters, opening two false color images and a histogram, changing the ordinate of the second false color image into FSC-H, and changing the abscissa of the histogram into PE-A;
s3, placing the uniformly mixed sample tube filled with the detection liquid into a sample loading groove, naming and operating; the first pseudo-color map is provided with a gate P1, enclosing a main body cell group; adjusting the second pseudo-color image to display cells in a P1 gate, arranging a gate P2, removing adhesion cells, adjusting the histogram to display cells in a P2 gate, adjusting the parameters of the histogram, and clicking to record;
s3, sequentially loading samples and detecting;
after completion of the test at S4, analysis was performed using Flowjo analysis software.
The sample loading speed of the step S3 is 5-25 mul/min; preferably, the sample loading speed of the step S3 is 8-15 μ l/min; more preferably, the sample application rate of step S3 is 10 μ l/min.
In the research of the applicant, the enzyme solution and the staining solution with specific volume ratios are selected in the invention, although RNA digestion can be well realized, when the machine speed is excessively high, the detection is easy to see inaccurate, and a great deal of applicants find that the accuracy of the detection method is improved when the sample loading speed is selected to be 8-15 mu l/min, probably because RNA digestion is well realized by using the enzyme solution and the staining solution with specific ratios in the invention, and the numerical CV value is stable under the condition of the machine speed of 8-15 mu l/min, and the accuracy of the detection result is high.
In step S4,A streaming PE channel is set, and the abscissa FLZ-A: the PE-A range is set to 0-400K.
Has the advantages that:
the method can better detect the cell cycle of the mesenchymal stem cells by selecting and controlling the proportion of the enzyme solution and the staining solution and simultaneously controlling the sample loading speed, and has better precision and accuracy.
Detailed Description
In order to better understand the technical solution, the technical solution will be described in detail with reference to specific embodiments; in addition, the starting materials used are all commercially available, unless otherwise specified.
Example 1
A high-accuracy cell cycle detection method comprises the following steps:
(1) cell thawing: placing the freezing tube filled with the frozen material in a water bath kettle at 37 deg.C for 2min to melt, centrifuging, and resuspending in MM3 complete culture medium to obtain resuscitated cells;
(2) cell inoculation: inoculating the revived cells into a culture bottle to obtain inoculated cells with the fusion degree of 80%;
(3) cell fixation: collecting 4X 10 6 Washing the inoculated cells for 2 times by using PBS buffer solution, centrifuging, removing supernatant, adding 100 mu l of PBS buffer solution into the precipitate, resuspending the precipitate by using 1mL of cell fixing solution, and standing the cell in a refrigerator at 4 ℃ overnight;
(4) enzymolysis: centrifuging the fixed cells, washing by using PBS buffer solution, removing supernatant, re-suspending the precipitate by using 100 mu l of PBS buffer solution to obtain cell suspension, and then adding 3 mu l of RNase enzyme solution into the cell suspension to obtain enzymatic hydrolysate;
(5) dyeing: adding 25ug/mL PI dye solution into the enzymatic hydrolysate, mixing, and incubating in dark to obtain detection solution;
(6) and (3) computer detection: detecting and analyzing the detection liquid on a machine by using a flow cytometry analyzer;
the frozen stock in the step (1) is frozen stock solution and mesenchymal stem cells; the mesenchymal stem cells are P5 generation human umbilical cord mesenchymal stem cells; the volume of MM3 complete medium was 5 times the volume of the frozen stock in the frozen stock tube;
the inoculation density of the revival cell inoculation in the step (2) is 6000/cm 2 ；
The volume concentration of the fixing solution is 70%; the stationary liquid is an ethanol water solution precooled at the temperature of minus 20 ℃;
in the step (5), the light-proof temperature is 23 ℃, and the light-proof time is 30 min; the volume ratio of the enzyme solution to the PI dye solution is 1.5: 100, respectively;
the method comprises the following steps of computer detection and analysis of the flow cytometry analyzer:
s1, opening the flow cytometry, newly building an experimental method, and cleaning and preheating the instrument;
s2 setting parameters, opening two false color images and a histogram, changing the ordinate of the second false color image into FSC-H, and changing the abscissa of the histogram into PE-A;
s3, placing the uniformly mixed sample tube filled with the detection liquid into a sample loading groove, naming and operating; the first pseudo-color map is provided with a gate P1, enclosing a main body cell group; adjusting the second pseudo-color image to display cells in a P1 gate, arranging a gate P2, removing adhesion cells, adjusting the histogram to display cells in a P2 gate, adjusting the parameters of the histogram, and clicking to record;
s3, sequentially loading samples and detecting;
after the detection of S4 is finished, Flowjo analysis software is used for analysis;
the sample loading speed of the step S3 is 10 mu l/min; in step S4,A streaming PE channel is set, and the abscissa FLZ-A: the PE-A range is set to 0-400K.
According to the detection steps, two different operators in a laboratory detect the same batch of the P5 generation human umbilical cord mesenchymal stem cells at the same time, each group of tests is repeated for 6 times, and the test results are shown in Table 1:
TABLE 1
According to the detection steps, the same operator detects the same batch of P5 generation human umbilical cord mesenchymal stem cells at different times, and the test results of each group of tests are repeated for 6 times as shown in Table 2:
TABLE 2
As is clear from the test results in tables 1-2, the RSD values between the measurement values of the cell cycles G0/G1, S and G2/M obtained by different operators at the same time and different times by the same operator are all less than or equal to 10%, indicating that the measurement values obtained by different operators in the analysis method have good intermediate precision.
Example 2
(1) Cell thawing: placing the freezing tube filled with the frozen material in a water bath kettle at 37 deg.C for 2min to melt, centrifuging, and resuspending in MM3 complete culture medium to obtain resuscitated cells;
(2) cell inoculation: inoculating the revived cells into a culture bottle to obtain inoculated cells with the fusion degree of 80%;
(3) cell fixation: collecting 4X 10 6 Washing the inoculated cells for 2 times by using PBS buffer solution, centrifuging, removing supernatant, adding 100 mu l of PBS buffer solution into the precipitate, resuspending, fixing the cells by using 1mL of cell fixing solution, and standing overnight in a refrigerator at 4 ℃;
(4) enzymolysis: centrifuging the fixed cells, washing by using PBS buffer solution, removing supernatant, re-suspending the precipitate by using 100 mu l of PBS buffer solution to obtain cell suspension, and then adding 3 mu l of RNase enzyme solution into the cell suspension to obtain enzymatic hydrolysate;
(5) dyeing: adding 25ug/mL PI dye solution into the enzymatic hydrolysate, mixing, and incubating in dark to obtain detection solution;
(6) and (3) computer detection: detecting and analyzing the detection liquid on a machine by using a flow cytometry analyzer;
the frozen stock in the step (1) is frozen stock solution and mesenchymal stem cells; the mesenchymal stem cells are P5 generation human umbilical cord mesenchymal stem cells; the volume of MM3 complete medium was 5 times the volume of the frozen stock in the frozen stock tube;
the inoculation density of the revived cell inoculation in the step (2) is 6000 cells/cm 2 ；
The volume concentration of the fixing solution is 70%; the stationary liquid is an ethanol aqueous solution pre-cooled at the temperature of minus 20 ℃;
in the step (5), the light-proof temperature is 23 ℃, and the light-proof time is 30 min; the volume ratio of the enzyme solution to the PI dye solution is 0.3: 100, respectively;
the method comprises the following steps of computer detection and analysis of the flow cytometry analyzer:
s1, opening the flow cytometry, newly building an experimental method, and cleaning and preheating the instrument;
s2 setting parameters, opening two false color images and a histogram, changing the ordinate of the second false color image into FSC-H, and changing the abscissa of the histogram into PE-A;
s3, placing the uniformly mixed sample tube filled with the detection liquid into a sample loading groove, naming and operating; the first pseudo-color map is provided with a gate P1, enclosing a main body cell group; adjusting the second pseudo-color image to display cells in a P1 gate, arranging a gate P2, removing adhesion cells, adjusting the histogram to display cells in a P2 gate, adjusting the parameters of the histogram, and clicking to record;
s3, sequentially loading samples and detecting;
after completion of the test at S4, analysis was performed using Flowjo analysis software.
The sample loading speed of the step S3 is 60 mu l/min; in step S4,A streaming PE channel is set, and the abscissa FLZ-A: the PE-A range is set to 0-400K.
According to the detection steps, the same operator detects the same batch of P5 generation human umbilical cord mesenchymal stem cells at different times, and the test results of each group of tests are repeated for 6 times as shown in Table 2:
TABLE 2
Claims (10)
1. A method for detecting a cell cycle with high accuracy, comprising the steps of:
(1) cell thawing: taking a freezing tube filled with the frozen material for thawing, centrifuging and resuspending to obtain resuscitated cells;
(2) cell inoculation: inoculating the revived cells into a culture bottle to obtain inoculated cells;
(3) cell fixation: collecting inoculated cells, washing, centrifuging, removing supernatant, precipitating and resuspending, and fixing the cells by using a cell fixing solution;
(4) enzymolysis: preparing the fixed cells into cell suspension, and then adding an enzyme solution into the cell suspension to obtain enzymatic hydrolysate;
(5) dyeing: adding a staining solution into the enzymatic hydrolysate, mixing uniformly, and incubating in a dark place to obtain a detection solution;
(6) and (3) computer detection: detecting and analyzing the detection liquid on a machine by using a flow cytometry analyzer;
resuspending in step (3) using PBS buffer; in step (3), PBS buffer used for resuspension: inoculating cells: fixative 100 μ l: 2.5X 10 6 -7×10 6 The method comprises the following steps: 1 mL.
2. The method for detecting cell cycle with high accuracy according to claim 1, wherein the frozen stock in step (1) is frozen stock solution and mesenchymal stem cells; the mesenchymal stem cells are Pi-generation mesenchymal stem cells, and i is an integer more than or equal to 1.
3. The method for detecting a cell cycle with high accuracy as claimed in claim 1, wherein the mesenchymal stem cell is at least one selected from the group consisting of human umbilical cord mesenchymal stem cell, adipose mesenchymal stem cell, placenta mesenchymal stem cell, dental pulp mesenchymal stem cell.
4. The method for detecting a cell cycle with high accuracy as set forth in any one of claims 1 to 3, wherein the resuspension conditions in step (1) are: MM3 was resuspended in complete medium, the volume of MM3 complete medium was 5 times the volume of the frozen stock in the frozen tubes.
5. The method for detecting a cell cycle with high accuracy as set forth in claim 1, wherein the confluency of the seeded cells in the step (2) is 75 to 85%.
6. The method for detecting cell cycle with high accuracy as claimed in claim 1 or 5, wherein the inoculation density of the resuscitated cell in step (2) is 5000-7000 cells/cm 2 。
7. The method for detecting a cell cycle with high accuracy as set forth in claim 1, wherein the washing solution in the step (3) is PBS buffer; the volume concentration of the fixing liquid is 60-80%.
8. The method according to claim 1, wherein the staining solution is 20-30ug/mL of PI staining solution.
9. The method for detecting a cell cycle with high accuracy according to claim 1 or 8, wherein the volume ratio of the enzyme solution to the staining solution is (1-2): 100.
10. the method for detecting a cell cycle with high accuracy according to claim 1, wherein the light shielding temperature in the step (5) is 20 to 26 ℃ and the light shielding time is 20 to 40 min.
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| CN115372233A (en) * | 2022-10-25 | 2022-11-22 | 华夏源(上海)生命科技有限公司 | Method for detecting cell cycle of adipose-derived stem cells |
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| CN115372233A (en) * | 2022-10-25 | 2022-11-22 | 华夏源(上海)生命科技有限公司 | Method for detecting cell cycle of adipose-derived stem cells |
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