CN100432653C - Counting method for trace suspension tumor cell - Google Patents
Counting method for trace suspension tumor cell Download PDFInfo
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- CN100432653C CN100432653C CNB2005100281468A CN200510028146A CN100432653C CN 100432653 C CN100432653 C CN 100432653C CN B2005100281468 A CNB2005100281468 A CN B2005100281468A CN 200510028146 A CN200510028146 A CN 200510028146A CN 100432653 C CN100432653 C CN 100432653C
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Abstract
The present invention discloses a counting method for trace suspension tumor cells. A cell liquid to be measured is fully and uniformly mixed by a suction pipe, and 1 mu l of cell liquid is sucked from a middle layer. Liquid around a gun head is lightly effaced, a thin wall EP pipe is laid flatly on a desktop, 1 mu l of cell suspension liquid is rapidly added in a small pipe by the gun head to become a liquid droplet in a small dot shape, and a lid is covered on the pipe. After the pipe is placed for one to two minutes, all the cells in the liquid droplet are directly counted under an inverted microscope. The method has low requirements for devices and reagents, and general laboratory conditions can satisfy the requirements. In addition, the method can also dilute the cells according to an arbitrary proportion, can accurately count cells of various concentration levels and can adapt to the requirements of various experimental researches. The method can also be applied to a plurality of laboratory technologies, such as the clinical detection of tumor cells, the preparation of monoclonal antibodies, the PCR amplification of single cells, etc.
Description
Technical field
The present invention relates to the method for cell count in a kind of cytologic experiment, specifically a kind of method of counting of trace suspension tumor cell.
Background technology
Cell count is a basic fundamental of cytologic experiment, and it is to understand the cultured cell growth conditions, draws growth curve, measures the important means of material biological actions such as nutrient culture media, serum, medicine.Need to carry out cell count in the former generation of zooblast and the numerous technology such as cultivation, cell freezing and recovery that go down to posterity, vegetable cell carries out suspension cultured also needs to measure cell number.Method for cell count commonly used has classical counting method, electronic cell calculating instrument counting method, MTT colourimetry or the like.In addition, also have some method of counting of improveing at some specific sample.Classical approach carries out Cytometric method with blood counting chamber exactly, and it is to clean tally and special cap slide with alcohol earlier, wipes away dried subsequently with silk.Last covering slide, with suction pipe cell liquid to be measured is beaten evenly, the middle level is drawn 10 μ l cell liquid and is put on the cover glass edge, treat that capillary action enters in the space between cover plate and the slide liquid, do not overflow cover glass, do not want very few or the band bubble yet, placed 1～2 minute, with the cell number in four jiaos of big grids of 10X object lens observation tally.When cell was pressed center line, only meter left side and top person disregarded right side and below person.After all having counted,, use 70% alcohol flushing again, blot with lens wiping paper with distilled water flushing blood cell counting plate and cover glass.Calculate tumor cell number by following formula: contain cell number=(4 big lattice cell number sum/4) * 10 in every ml stoste
4* extension rate.Up to the present the most classical counting technology of blood counting chamber counting method, it can be used for blood count, cultured cells and need be at the various samples of microscopically counting.But when being to use classical counting method limitation is arranged also, cell concentration must be greater than 2 * 10 when promptly counting
4Individual/ml, so classical approach is not suitable for concentration less than 2 * 10
4The cell liquid counting of individual/ml.As when carrying out the PCR operation, last cell liquid often has only several microlitres, and cell liquid concentration is very low again.
Summary of the invention
Technical matters to be solved by this invention provides a kind of method of counting of quick, effective trace suspension tumor cell, to be fit to the needs of various experimental studies.
Technical matters to be solved by this invention can realize by the following technical solutions.
A kind of method of counting of trace suspension tumor cell, it is fully to beat even cell liquid to be measured with suction pipe, draws the cell liquid of 1 μ l in the middle level with the rifle head; Wipe rifle head liquid on every side gently away; Thin-walled EP pipe is lain in desktop, and the rifle head adds 1 μ l cell suspension apace in thin-walled EP pipe, make its drop that becomes a roundlet point-like, cover lid; Place after 1～2 minute the whole cells under inverted microscope in the direct census drop.
Method of the present invention is very low to the requirement of equipment, reagent, and general laboratory condition can meet the demands.In addition, this method also can be pressed cell the arbitrary proportion dilution, to the cell number average energy accurate counting of various concentration levels, is fit to the needs of various experimental studies.This method can also be applied in as many laboratory techniques such as clinical detection tumour cell, preparation monoclonal antibody, unicellular pcr amplifications at other.
Embodiment
Relatively further specify the present invention below in conjunction with the method for counting of classical approach and trace suspension tumor cell of the present invention.
1 materials and methods
1.1 material
Select for use Chinese Academy of Sciences's Shanghai cell the K562 tumour cell, with containing the RPMI1640 nutrient solution of 10%FCS, place 37 ℃, cultivate in the 5%CO2 saturated humidity incubator.During experiment,, get its mean value (with it as normal concentration) routinely with blood counting chamber counting 3 times.The cell liquid of getting 5 μ l diluted 1: 5,1: 10,1: 15,1: 20,1: 25,1: 30,1: 40,1: 50 with the PBS balanced salt solution, get the sample of known variable concentrations tumour cell, get above sample counting cells with classical approach and method of the present invention, its mean value is got in every kind of method average test 3 times.
1.2 instrument
Blood counting chamber, counter, application of sample rifle, aseptic straw, thin-walled EP pipe, ordinary optical microscope, inverted microscope.
1.3 method
1.3.1 classical counting method: with alcohol cleaning tally and special cap slide, wipe away dried subsequently with silk earlier.Last covering slide, with suction pipe cell liquid to be measured is beaten evenly, the middle level is drawn 10 μ l cell liquid and is put on the cover glass edge, treat that capillary action enters in the space between cover plate and the slide liquid, do not overflow cover glass, do not want very few or the band bubble yet, placed 1～2 minute, with the cell number in four jiaos of big grids of 10X object lens observation tally.When cell was pressed center line, only meter left side and top person disregarded right side and below person.After all having counted,, use 70% alcohol flushing again, blot with lens wiping paper with distilled water flushing blood cell counting plate and cover glass.Calculate tumor cell number by following formula: contain cell number=(4 big lattice cell number sum/4) * 10 in every ml stoste
4* extension rate.
1.3.2 the method for counting of trace suspension tumor cell of the present invention: fully beat even cell liquid to be measured with suction pipe earlier, then draw the cell liquid of 1 μ l fast in the middle level with the rifle head.Wipe rifle head liquid on every side gently away with silk.0.2cm thin-walled EP pipe is lain in desktop, and the rifle head adds 1 μ l cell suspension apace in thin-walled EP pipe, make its drop that becomes a roundlet point-like, cover lid.Place after 1～2 minute the whole cells under inverted microscope in the direct census drop.
1.3.3 statistical method, the gained data are so that (x ± s) expression, front and back numerical value difference is relatively with t check, straight-line regression, correlation analysis.
2 results
2.1 undiluted cell, two kinds of method of counting of the method for counting of classical approach and trace suspension tumor cell of the present invention relatively.It the results are shown in Table 1.
Two kinds of method of counting results that table 1 is undiluted are (unit: * 10 relatively
4Individual/ml)
By calculating t=2.138, table look-up and learn t
0.05＞t, P＞0.05, count results does not have significant difference.Show this two kinds of method count results unanimities.In some cases, can substitute classical approach with the method for counting of trace suspension tumor cell of the present invention and carry out cell count.
2.2 the cell after the dilution, two kinds of method of counting of the method for counting of classical approach and trace suspension tumor cell of the present invention relatively.
Cell concentration is 67 * 10 in the undiluted sample
4/ ml measures the sample of same extension rate with the method for the method of counting counting cells of classical approach and trace suspension tumor cell of the present invention, and it the results are shown in Table 2.
2.2.1 with classical counting method counting cells concentration value is X, micro-counting method count results value is Y, and linear relationship between the two is: correlation coefficient r=0.998461.When tabling look-up P=0.01, r
0.01＜r, correlation it very is remarkable, for tetanic line is correlated with.
2.2.2 the extension rate of counting with the method for counting of classical approach and trace suspension tumor cell of the present invention is an independent variable respectively, and each concentration gradient is carried out linear regression analysis, the result is:
(1) classical counting method correlation coefficient r=-0.89462; The result shows that correlativity is stronger between extension rate and the cell concentration, for negative tetanic line is correlated with.Regression equation: y=124.0167-4.07476x;
(2) micro-counting method correlation coefficient r=-0.80930; The result shows that correlativity is stronger between extension rate and the cell concentration, for negative tetanic line is correlated with.Regression equation: y=92.54143-2.03931x;
After table 2 dilution, two kinds of method of counting results compare (unit: individual/μ l)
From above data as can be seen two kinds of method count results be more or less the same, its result shows that classical counting method is in cell concentration＜2 * 10
4Individual/during ml, its actual measured value is " 0 ".The method of counting of trace suspension tumor cell of the present invention is in cell concentration＜2 * 10
4Individual/during ml, its measured value and expectation value are close.
Can learn that from above result the dilution operation can not influence the result of the method for counting of trace suspension tumor cell of the present invention.Whether no matter dilution process arranged, and all the result with this basic assay method of classical counting method is close for the result that micromethod is measured.When cell liquid concentration is lower than 2 * 10
4Individual/during ml, classical approach counting measured value is " 0 ", and the result of micromethod counting still is close with expectation value.
So,, be lower than 2 * 10 in cell liquid concentration though the degree of accuracy of the method for counting of trace suspension tumor cell of the present invention does not have classical approach good
4Individual/as during ml, can to use micromethod to substitute classical counting method.When operating, note making cell to be dispersed into individual cells, before the sampling counting as far as possible, fully the mixing cell suspending liquid when serial sampling is counted, should be noted this point especially, otherwise can influence the cell count result, very big error appears in count results before and after making.During the microscopically counting, run into the cell mass that 2 above cells are formed, should calculate,, illustrate that dispersion is insufficient as cell mass＞10% by individual cells.
The principle of the method for counting counting of trace suspension tumor cell of the present invention is exactly the cell liquid certain volume, directly calculates its number at microscopically, and the result that utilization is drawn extrapolates the cell quantity in every ml cells liquid.Micromethod is measured and is analyzed and not only is confined to a certain single method, and micromethod also can be applied to a lot of fields in fact.
The method of counting of trace suspension tumor cell of the present invention can be applied to the quantity of clinical detection tumour cell.Malignant tumour is one of principal disease that threatens at present human health and life, and people are exploring the new way and the new method of diagnosing tumor treatment always.Tumour cell constantly divides, and forms new tumour cell, and by tissue infiltration and its place's transfer towards periphery of former position, this transfer such as uncontrollable will further be invaded critical organ and depletion be caused death at last with causing.Have experiment to show, when a small amount of oncocyte shifts, can form the lethal tumour, a large amount of identical tumour cells shift then mostly is ostracised.So when the several tumour cells that have only minority begin to shift, can not find especially, often cause clinical false judgment, forfeiture treatment opportunity with general imaging diagnosis method.We can consider to utilize the method for counting of SABC and trace suspension tumor cell of the present invention to detect the quantity of tumour cell, obtain single tumour cell then and study.
Specific practice is: in the clinical detection, contain 7000-10000 cell usually in the 1 μ l blood.In the blood of 1 μ l, add antibody, antigen, by the antibody antigen association reaction, isolated several cells that contain cancer antigen, this moment, classical approach can not counted the result certainly.In this case, can determine comedocarcinoma anti-cell content with the method for counting of trace suspension tumor cell of the present invention, extrapolating whole body has several cancer anti-cells.The result that this method is measured can be used as postoperative and follows up a case by regular visits to, the index of monitoring tumor recurrence, transfer.
Method of counting with trace suspension tumor cell of the present invention can also be produced out a cell from a large amount of cell suspensions.According to the research prompting of classics, the B emiocytosis antibody in the body, the immunoglobulin (Ig) that each B emiocytosis is a type.The antigen that exists in the nature reaches 10
8More than, human body is dealt with such complex environment in order to survive, must set up a huge antibody library, so the antibody that produces of each B cell we can prepare monoclonal antibody with micromethod.MONOCLONAL ANTIBODIES SPECIFIC FOR relates generally to technology such as animal immune, Fusion of Cells, selection cultivation, cloning.Wherein individual cells is isolated in cloning exactly, makes it pass through division growth and obtains the very all processes of the cell colony of homogeneous of a heredity.After cloning for several times, can set up monoclonal cell system.We can use micromethod to carry out cloning.Cell suspension through repeatedly dilution, may only be contained the concentration of 1 cell, and makes cell proliferation in every hole of 96 well culture plates, thereby obtain monoclonal antibody clone.
Unicellular gene diagnosis is a new and high technology, and with development of biology, increasing new technology is applied to this field, and more genetic disease is effected a radical cure.Similarly, can use the method for counting of trace suspension tumor cell of the present invention from about 10
6Take out a cell in the cell liquid of individual/ml and carry out unicellular pcr amplification.
Claims (2)
1, a kind of method of counting of trace suspension tumor cell, it is fully to beat even cell liquid to be measured with suction pipe, draws the cell liquid of 1 μ l in the middle level with the rifle head; Wipe rifle head liquid on every side gently away; Thin-walled EP pipe is lain in desktop, and the rifle head adds 1 μ l cell suspension apace in thin-walled EP pipe, make its drop that becomes a roundlet point-like, cover lid; Place after 1～2 minute the whole cells under inverted microscope in the direct census drop.
2, the method for counting of a kind of trace suspension tumor cell according to claim 1 is characterized in that: during the microscopically counting, run into the cell mass that 2 above cells are formed, press individual cells and calculate, as cell mass＞10%, illustrate that dispersion is insufficient.
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| CNB2005100281468A CN100432653C (en) | 2005-07-26 | 2005-07-26 | Counting method for trace suspension tumor cell |
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| CN109055213A (en) * | 2018-08-14 | 2018-12-21 | 深圳职业技术学院 | A kind of production method of simple Micro-CPE neutralization test and the device and the device of counting |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN2377532Y (en) * | 1999-06-10 | 2000-05-10 | 中国人民解放军第二军医大学 | Microfluorecyte counting plate |
| WO2003001904A1 (en) * | 2001-06-27 | 2003-01-09 | Delaval Holding Ab | A method of and an apparatus for somatic cell count |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN2377532Y (en) * | 1999-06-10 | 2000-05-10 | 中国人民解放军第二军医大学 | Microfluorecyte counting plate |
| WO2003001904A1 (en) * | 2001-06-27 | 2003-01-09 | Delaval Holding Ab | A method of and an apparatus for somatic cell count |
Non-Patent Citations (4)
| Title |
|---|
| 肿瘤细胞计数--单波长噻唑蓝比色分析法. 焦顺昌，赵东海，黄昌霞，李求是.肿瘤，第18卷第5期. 1993 |
| 肿瘤细胞计数--单波长噻唑蓝比色分析法. 焦顺昌，赵东海，黄昌霞，李求是.肿瘤，第18卷第5期. 1993 * |
| 采用视频***的细胞原位计数法. 贾倞，王坤，迟永春，赵天德.中日友好医院学报，第15卷第2期. 2001 |
| 采用视频***的细胞原位计数法. 贾倞，王坤，迟永春，赵天德.中日友好医院学报，第15卷第2期. 2001 * |
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