CN104694617A - Cell proliferation rate detection method and application - Google Patents
Cell proliferation rate detection method and application Download PDFInfo
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- CN104694617A CN104694617A CN201510119745.4A CN201510119745A CN104694617A CN 104694617 A CN104694617 A CN 104694617A CN 201510119745 A CN201510119745 A CN 201510119745A CN 104694617 A CN104694617 A CN 104694617A
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Abstract
The invention discloses a cell proliferation rate detection method and application, belonging to the technical field of cell engineering. The method comprises the steps: respectively sampling at different time-points of culturing cells; treating the obtained samples by virtue of colchicines and fixing cells by means of ethyl alcohol after treatment; detecting a cell cycle for the fixed cells in use of a flow cytometry, and meanwhile, detecting the cell cycle of a blank control group which is not subjected to the treatment of colchicines; calculating cell proliferation rates, drawing a cell growth curve and determining the proliferation rate of to-be-detected cells by means of the cell growth curve. In the method, on the basis that the precision of the flow cytometry is guaranteed, the detection cost is reduced greatly and the application range is expanded.
Description
Technical field
The present invention relates to a kind of measuring method and application of cell proliferation rate, belong to cell engineering field.
Background technology
It is the most basic experimental technique of cell biology that cell proliferation rate measures, traditional chemical process precision is not high, existing cells were tested by flow cytometry technology is (as BrdU method, PCNA method etc.), although solve the problem of measuring accuracy, but BrdU method and PCNA method all depend on the antibody of special marking, these antibody are expensive and have species specificity, and this just greatly limit the range of application of these two kinds of methods.
Summary of the invention
For solving the problem, the invention provides a kind of measuring method of cell proliferation rate, the technical scheme taked is as follows:
The object of the present invention is to provide a kind of measuring method of cell proliferation rate, the method samples respectively in the different time points of culturing cell, colchicine is utilized to process institute's sample thief, ethanol fixed cell is utilized after process, recycling flow cytometer carries out cell cycle detection to fixed cell, detect the cell cycle of the blank group without colchicine process simultaneously, calculate cell proliferation rate, draw cell growth curve, recycling cell growth curve determines the multiplication rate of cell to be measured.
The step of described method is as follows:
1) containing 5%CO
2culturing cell in incubator, and sample in the different time points of Growth of Cells;
2) colchicine is utilized to step 1) cell that samples processes, and process terminates rear stand for standby use, arranges the blank group without colchicine process simultaneously;
3) ethanol is utilized to step 2) processing sample of gained and control sample be fixed process, obtains fixed cell;
4) cells were tested by flow cytometry step 3 is utilized) cell cycle of the fixed cell of gained, and calculate the multiplication rate of cell, draw cell growth curve;
5) step 4 is utilized) cell growth curve of gained measures the multiplication rate of cell to be measured.
Preferably, step 1) described cell is pilose antler mescenchymal stem cell; Described different time points samples at 12h, 24h, 36h, 48h, 60h and 72h of cell cultures; Described sampling gets 10 with the culture dish of diameter 100mm
6individual cell.
Preferably, step 2) described colchicine process utilizes the colchicine of final concentration 80-120 μ g/ml to process cell.
More preferably, described colchicine process utilizes the colchicine of final concentration 100 μ g/ml to process cell.
Preferably, step 2) described standing, the time left standstill is 6h.
Preferably, step 3) described fixing process, be utilize 70% ethanol fixed cell.
Preferably, step 4) describedly utilize the cells were tested by flow cytometry cell cycle, the cell sample PBS fixed washs, after PI dyeing, with flow cytometry analysis G2/M phase cell proportion.
The concrete steps of described method are as follows:
1) containing 5%CO
2cultivate pilose antler mescenchymal stem cell in incubator, and sample at 12h, 24h, 36h, 48h, 60h and 72h of Growth of Cells;
2) colchicine of final concentration 100 μ g/ml is utilized to step 1) cell sample that obtains processes, leaves standstill 6h, arrange the blank group without colchicine process simultaneously after process;
3) ethanol of 70% is utilized to step 2) processing sample of gained and control sample be fixed process, obtains fixed cell;
4) cells were tested by flow cytometry step 3 is utilized) cell cycle of the fixed cell of gained, and calculate the multiplication rate of cell, draw cell growth curve;
5) step 4 is utilized) cell growth curve of gained measures the multiplication rate (as Fig. 1) of cell to be measured.
Described either method is for measuring multiplication rate and the growth curve of cell.
The beneficial effect that the present invention obtains is as follows:
The present invention selects regular growth to divide blocker--and colchicine blocks the cell cycle, and compared with traget antibody, colchicine is cheap, and does not have species specificity.Utilize the distribution in cells were tested by flow cytometry sample cell cycle, compare the changes delta G2/M that colchicine adds the front and back cell cycle, assessment cell proliferation rate, draw growth curve by the Δ G2/M of METHOD FOR CONTINUOUS DETERMINATION different time points.By with the comparing of common detection methods mtt assay and cell counting, the result of the result cell counting of Δ G2/M is consistent, and this method is more accurate than mtt assay.
Compared with prior art, the present invention is on the basis ensureing flow cytometer precision, greatly reduces cost of determination, has widened range of application.
Accompanying drawing explanation
Fig. 1 draws growth curve according to Δ G2/M.
Fig. 2 is the growth curve drawn according to the inventive method, mtt assay and cell counting;
(AP: pilose antler mescenchymal stem cell, FP: face's periosteum cell, 293T:293T cell).
Embodiment
Below in conjunction with specific embodiment, the present invention will be further described, but the present invention is not by the restriction of embodiment.
The material that following examples use, reagent, instruments and methods, without specified otherwise, be this area conventional material, reagent, instruments and methods, all obtain by commercial channel.
Embodiment 1
Present embodiments provide and utilize mtt assay and cell counting to draw the method for cell growth curve, step is as follows:
1) conventional CO is utilized
2incubator culturing cell, and sample at 12h, 24h, 36h, 48h, 60h and 72h of Growth of Cells;
Mtt assay: the MTT adding final concentration 20ug/ml at 12h, 24h, 36h, 48h, 60h and 72h of Growth of Cells, after 2h, removes nutrient solution, adds DMSO, measure OD value after mixing.
Cell counting: at 12h, 24h, 36h, 48h, 60h and 72h of Growth of Cells, with pancreatin by cell suspension, counts with blood counting chamber.
2) according to the result of above-mentioned two kinds of method gained, and calculate the multiplication rate of cell, draw growth curve (growth curve as in the middle of Fig. 2 and right side).
Embodiment 2
1) utilize ordinary method to cultivate mescenchymal stem cell, and sample at the 24h of Growth of Cells;
2) colchicine of final concentration 100 μ g/ml is utilized to step 1) cultured cells sample processes, and leave standstill 2h, 4h, 6h, 8h, 10h, 12h, 14h after process, the blank group without colchicine process is set simultaneously;
3) ethanol of 70% is utilized to step 2) processing sample of gained and control sample be fixed process, obtains fixed cell;
4) cells were tested by flow cytometry step 3 is utilized) cell cycle of the fixed cell of gained, and calculate the multiplication rate of cell;
5) step 4 is utilized) multiplication rate of the cell of gained, draw growth curve.
Result shows, the time, too short Δ G2/M was too little, and overlong time Δ G2/M slowdown in growth even declines, and finally determines that the optimum handling time is 4-8h.
Embodiment 3
1) utilize ordinary method to cultivate mescenchymal stem cell, and sample at 12h, 24h, 36h, 48h, 60h and 72h of Growth of Cells;
2) colchicine of final concentration 80 μ g/mL, 100 μ g/mL and 120 μ g/mL is utilized to step 1) cultured cells sample processes, and leave standstill 6h after process, the blank group without colchicine process is set simultaneously;
3) ethanol of 70% is utilized to step 2) processing sample of gained and control sample be fixed process, obtains fixed cell;
4) cells were tested by flow cytometry step 3 is utilized) cell cycle of the fixed cell of gained, the sample fixed with PBS washing 2 times, PI lucifuge dyeing 15min, upper machine measures the cell cycle, and calculate the multiplication rate parameter Δ G2/M of cell, draw cell growth curve;
Result shows, the growth curve of mensuration meets the growth rhythm of general cell, and under the colchicine process of three kinds of concentration, cell growth curve is without significant difference.
Embodiment 4
The rate of propagation of the more different cell of unitary determination
1) utilize ordinary method to cultivate mescenchymal stem cell, 293T cell, adult periosteum cell, and sample at the 24h of Growth of Cells; Simultaneously according to embodiment 1) in conventional mtt assay measure the rate of propagation of three kinds of cells;
2) colchicine of final concentration 100 μ g/mL is utilized to step 1) cultured cells sample processes, and leave standstill 6h after process, the blank group without colchicine process is set simultaneously;
3) ethanol of 70% is utilized to step 2) processing sample of gained and control sample be fixed process, obtains fixed cell;
4) cells were tested by flow cytometry step 3 is utilized) cell cycle of the fixed cell of gained, with the fixing sample of PBS washing 2 times, PI lucifuge dyeing 15min, upper machine measures the cell cycle, and calculates the multiplication rate parameter Δ G2/M of cell.
Embodiment 5
1) utilize ordinary method to cultivate mescenchymal stem cell, add the growth of rottlerin (20 μMs) interference cell in cell culture medium, and sample at 12h, 24h, 36h, 48h, 60h and 72h of Growth of Cells; Simultaneously by embodiment 1) mtt assay mensuration cell proliferation rate;
2) final concentration 100 μ g/mL step 1 is utilized) cultured cells sample processes, and leave standstill 6h after process, the blank group without colchicine process is set simultaneously;
3) ethanol of 70% is utilized to step 2) processing sample of gained and control sample be fixed process, obtains fixed cell;
4) cells were tested by flow cytometry step 3 is utilized) cell cycle of the fixed cell of gained, the sample fixed with PBS washing 2 times, PI lucifuge dyeing 15min, upper machine measures the cell cycle, and calculate the multiplication rate parameter Δ G2/M of cell, draw cell growth curve.
Embodiment 6
1) utilize ordinary method to cultivate pilose antler mescenchymal stem cell, and sample at 12h, 24h, 36h, 48h, 60h and 72h of Growth of Cells;
2) colchicine of final concentration 100 μ g/mL is utilized to step 1) cultured cells sample processes, and leave standstill 6h after process, the blank group without colchicine process is set simultaneously;
3) ethanol of 70% is utilized to step 2) processing sample of gained and control sample be fixed process, obtains fixed cell;
4) cells were tested by flow cytometry step 3 is utilized) cell cycle of the fixed cell of gained, the sample fixed with PBS washing 2 times, PI lucifuge dyeing 15min, upper machine measures the cell cycle, and calculate the multiplication rate parameter Δ G2/M of cell, draw cell growth curve; Fixing sample PCNA antibody test positive rate, concrete steps: (mouse monoclonal antibody PCNA 2h, the anti-2h of sheep anti mouse fluorescent mark two, upper machine testing positive rate).
Embodiment 7
The present embodiment compares the technique effect of method described in embodiment 1 and embodiment 4-6, as shown in table 1.
Table 1 embodiment 1 and embodiment 4-6 method and technology effect comparison
As can be seen from Table 1, the accuracy rate utilizing method described in the application to measure in embodiment 4-6 is all better than MTT method and cell counting, and the result measured is very stable, and method circulation ratio is fine.The cost utilizing method of the present invention to measure cell proliferation speed is especially only 8.33% of PCNA method cost.As can be seen from the result of embodiment 5, when there is the interference of medicine rottlerin, the accuracy rate of technical scheme provided by the present invention is far away higher than mtt assay.Embodiment 6 can find out that application antibody method is suitable with present method accuracy rate, but expense is far beyond technical scheme provided by the present invention.
Although the present invention with preferred embodiment openly as above; but it is also not used to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; can do various change and modification, what therefore protection scope of the present invention should define with claims is as the criterion.
Claims (10)
1. the measuring method of a cell proliferation rate, it is characterized in that, sample respectively in the different time points of culturing cell, utilize colchicine to process institute's sample thief, utilize ethanol fixed cell after process, recycling flow cytometer carries out cell cycle detection to fixed cell, detect the cell cycle of the blank group without colchicine process simultaneously, calculate cell proliferation rate, draw cell growth curve, recycling cell growth curve determines the multiplication rate of cell to be measured.
2. method described in claim 1, is characterized in that, step is as follows:
1) 5%CO is being contained
2culturing cell in incubator, and sample in the different time points of Growth of Cells;
2) colchicine is utilized to step 1) cell that samples processes, and process terminates rear stand for standby use, arranges the blank group without colchicine process simultaneously;
3) ethanol is utilized to step 2) processing sample of gained and control sample be fixed process, obtains fixed cell;
4) cells were tested by flow cytometry step 3 is utilized) cell cycle of the fixed cell of gained, and calculate the multiplication rate of cell, draw cell growth curve;
5) step 4 is utilized) cell growth curve of gained measures the multiplication rate of cell to be measured.
3. method described in claim 2, is characterized in that, step 1) described cell is pilose antler mescenchymal stem cell; Described different time points samples at 12h, 24h, 36h, 48h, 60h and 72h of cell cultures; Described sampling gets 10 with the culture dish of diameter 100mm
6individual cell.
4. method described in claim 2, is characterized in that, step 2) described colchicine process utilizes the colchicine of final concentration 80-120 μ g/ml to process cell.
5. method described in claim 4, is characterized in that, described colchicine process, is to utilize the colchicine of final concentration 100 μ g/ml to process cell.
6. method described in claim 2, is characterized in that, step 2) described standing, the time left standstill is 6h.
7. method described in claim 2, is characterized in that, step 3) described fixing process, be utilize 70% ethanol fixed cell.
8. method described in claim 2, is characterized in that, step 4) describedly utilize the cells were tested by flow cytometry cell cycle, the cell sample PBS fixed washs, after PI dyeing, with flow cytometry analysis G2/M phase cell proportion.
9. method described in claim 2, is characterized in that, concrete steps are as follows:
1) containing 5%CO
2cultivate pilose antler mescenchymal stem cell in incubator, and sample at 12h, 24h, 36h, 48h, 60h and 72h of Growth of Cells;
2) colchicine of final concentration 100 μ g/ml is utilized to step 1) cell sample that obtains processes, leaves standstill 6h, arrange the blank group without colchicine process simultaneously after process;
3) ethanol of 70% is utilized to step 2) processing sample of gained and control sample be fixed process, obtains fixed cell;
4) determination step 3) cell cycle of fixed cell of gained, and calculate the multiplication rate of cell, draw cell growth curve;
5) step 4 is utilized) cell growth curve of gained measures the multiplication rate of cell to be measured.
10. either method described in claim 1-9, is characterized in that, for measuring multiplication rate and the growth curve of cell.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111751522A (en) * | 2019-03-27 | 2020-10-09 | 中国科学院宁波工业技术研究院慈溪生物医学工程研究所 | Cell detection analyzer and cell proliferation information detection method |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1383452A (en) * | 1998-01-12 | 2002-12-04 | 新生物技术公司 | Compsns. and methods for treating cells having double minute DNA |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1383452A (en) * | 1998-01-12 | 2002-12-04 | 新生物技术公司 | Compsns. and methods for treating cells having double minute DNA |
Non-Patent Citations (2)
| Title |
|---|
| SCOTT CLARKE ET AL: "Breakthrough cell proliferation detection: click chemistry based labeling of nucleic acid analogue", 《CELLULAR AND MOLECULAR BIOLOGY》 * |
| SEVERINE PLANCHAIS，ET AL: "Chemical inhibitors: a tool for plant cell cycle studies", 《FEBS LETTERS》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111751522A (en) * | 2019-03-27 | 2020-10-09 | 中国科学院宁波工业技术研究院慈溪生物医学工程研究所 | Cell detection analyzer and cell proliferation information detection method |
| CN111751522B (en) * | 2019-03-27 | 2023-06-20 | 中国科学院宁波工业技术研究院慈溪生物医学工程研究所 | Cell detection analyzer and detection method of cell proliferation information |
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Application publication date: 20150610 |