CN102719398A - Novel efficient method for separating human T-Lymphocytes by immunomagnetic beads - Google Patents
Novel efficient method for separating human T-Lymphocytes by immunomagnetic beads Download PDFInfo
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- CN102719398A CN102719398A CN2011100797890A CN201110079789A CN102719398A CN 102719398 A CN102719398 A CN 102719398A CN 2011100797890 A CN2011100797890 A CN 2011100797890A CN 201110079789 A CN201110079789 A CN 201110079789A CN 102719398 A CN102719398 A CN 102719398A
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Abstract
The invention establishes an operation method in T-Lymphocytes culture in vitro by immunomagnetic bead separation technique to maximize the separation of human T-Lymphocytes from the same volume of human peripheral blood to obtain a lot of purified T-Lymphocytes and cover the shortage that lymphocytes can be only primary cultured but not be subcultured to the greatest extent. The method is simple and practicable.
Description
Technical field
The invention belongs to field of cell culture, be specifically related to human T lymphocyte's the sorting and the working method of magnetic activated cell seperation.
Background technology
The immunological magnetic bead sorting method is the advanced person's of widespread use in the present scientific research a cell separation technology.It is to combine with the monoclonal antibody specific that is connected magnetic bead through the cell-surface antigens molecule, and under the effect of externally-applied magnetic field, the feasible cell that links to each other with magnetic bead through monoclonal antibody is attracted and is trapped in the magnetic field; And do not have a cell of this surface antigen, because of can not combining, and can not in magnetic field, stop, whereby with cellular segregation with the specific monoclonal antibody that is connected magnetic bead.As the cell of suspension growth, the T lymphocyte can not be used the method for this separation of adherent sieve method attached cell.So sorting T lymphocytic method can be then mostly number average with immunomagnetic beads separation method or flow cytometry.But because the damage of flow cytometry pair cell is bigger, and may pollute the cell that sorts out, and can't carry out subsequent experimental, so this experiment still selects for use the immunomagnetic beads separation method as the lymphocytic prefered method of sorting T.
By directly take out in the body tissue or cell cultivate cry former be commissioned to train foster.The primary cultured cell isolated time is short, and is similar in proterties and the body, is applicable to research.The lymphocytic cultivation of T then belongs to former and is commissioned to train fosterly, owing to can't go down to posterity, viable cell quantity used in causing testing descends with the prolongation of experimental period gradually.So how can in the middle of the human peripheral of equal volume, maximize the sub-electing human T lymphocyte, prolong the lymphocytic survival time of T as far as possible, then become the hot technology that all are paid close attention in research lymphocyte cultivation field.
The objective of the invention is in the lymphocytic vitro culture technology of T, to make the experimenter obtain the T lymphocyte of large-scale purification.This method is simple to operate, feasible practicality.
Summary of the invention
Human T lymphocyte for the purifying that obtains greater amt overcomes the weakness that the T lymphocyte can't go down to posterity, and the present invention provides a kind of experimental implementation method, and making can the human T lymphocyte of sub-electing as much as possible in the middle of the human peripheral of equal volume.
The technical solution adopted for the present invention to solve the technical problems is: the present invention provides a kind of efficient immunological magnetic bead sorting human T lymphocyte's method, and in experiment, immunomagnetic beads method is in the lymphocytic process of immunomagnetic beads separation method sorting T; The utilization density gradient centrifugation is separated to and adds PBS and obtain with the mononuclearcell when being main cell suspension, and not with cover putting into after the magnetic pole 5 minutes do not taken out under the polar situation; With pipettor sucking-off cell suspension; And then the cell suspension of sucking-off sucked again in the test tube, put into magnetic pole 5 minutes, when then directly pouring out for the second time cell suspension; Renew PBS, repeat twice.Add the RPMI-1640 cell culture fluid, expect blue dyeing with platform, and carry out viable count, this moment, cell count was the lymphocytic quantity of isolated T.At first want action norm, then at test tube when magnetic pole is poured out PBS liquid, action should slowly also will link up, and has fallen and then need pause several seconds after the liquid.PBS changes at most three times, to reduce the lymphocytic loss of T.
The invention has the beneficial effects as follows that former how can in the middle of the human peripheral of equal volume, the maximization sub-elects the human T lymphocyte, prolong the lymphocytic survival time of T as far as possible, operation is simple.
Embodiment
1. materials and methods
1.1 research object
Normal adults (mainly screen healthy, the volunteer doner of blood of no disease) T lymphocyte.
1.2 material
1.3 the separation and Culture of human peripheral blood single nucleus cell
Human peripheral 15ml, anticoagulant heparin, with doubly dilution such as D-hank ' s buffered soln, the lymphocyte separation medium of choosing then separates (ratio of parting liquid and peripheral blood is 1: 1), leaves the heart 20 minutes with 2500.The tunica albuginea layer adds an amount of D-hank ' s buffered soln in the middle of drawing, and 1500 left the heart 10 minutes.With the washing of RPMI-1640 cell culture fluid once; Abandon supernatant; Add and to contain 2% foetal calf serum and PBS (phosphate buffered saline buffer) 8ml of 1mmol/L EDTA (YD 30), expect blue dyeing with platform, and carry out viable count; The viable cell number average is greater than more than 90%, and this moment, cell count was isolated mononuclearcell quantity.Selecting quantity is 1 * 10
7Individual mononuclearcell gets into next step experiment.
1.4 the lymphocytic separation and Culture of human peripheral T
At first, will before isolated total mononuclearcell be divided into A group and B group, every group contains 4ml PBS, makes the cell suspension in two 5ml round bottom polystyrene tubes contain the mononuclearcell of same order.1500 left the heart 5 minutes.Abandon supernatant, two pipes all add 100 μ l PBS piping and druming and process cell suspension, add the ratio of 100 μ lCD3 antibody then in every ml cell; Add corresponding CD3 antibody, the piping and druming mixing was hatched under the room temperature 15 minutes; Adding volume again is the half the immunomagnetic beads of CD3 antibody, incubated at room 10 minutes.Adding final volume is the PBS of 2.5ml.A organizes cell suspension, and not with cover putting into after the magnetic pole 5 minutes not taking out under the polar situation, poured out cell suspension, takes out test tube then and adds new PBS, triplicate so again.B organizes cell suspension, and not with cover putting into after the magnetic pole 5 minutes do not taken out under the polar situation; With pipettor sucking-off cell suspension, and then the cell suspension of sucking-off sucked again in the test tube, put into magnetic pole 5 minutes; When then directly pouring out cell suspension for the second time, renew PBS, repeat twice.Last two groups all add the RPMI-1640 cell culture fluid, expect blue dyeing with platform, and carry out viable count, and this moment, cell count was the lymphocytic quantity of isolated T.
1.5 data statistics
Mean relatively with the t check, represent with
by data between two groups.Adopt SPSS17.0 for WINDOWS software to analyze, insolation level is 0.05, when P＜0.05 explanation has statistical significance.
2. experimental result
Every group of isolated mononuclearcell all is 5 * 10
6Individual, the ratio of then isolated T lymphocyte quantity and mononuclearcell quantity, then between 12%～17%, B organizes then between 36%～60% the A group.
Table 1 liang group comparison
n=3 of isolated T lymphocyte quantity
*: compare t=3.337 P＜0.05 with the A group.
Claims (3)
1. an efficient immunological magnetic bead sorting human T lymphocyte method comprises when using density gradient centrifugation to be separated to adding PBS obtains with the mononuclearcell being the cell suspension of leading (liquid feeding for the first time), changes the PBS damping fluid repeatedly and is no more than three times.
2. according to claim 1, after it is characterized in that for the first time adding PBS, not with cover putting into after the magnetic pole 5 minutes; Do not taking out under the polar situation; With pipettor sucking-off cell suspension, and then the cell suspension of sucking-off sucked again in the test tube, put into magnetic pole 5 minutes.
3. according to claim 1, it is characterized in that after second and third time adds PBS, putting into magnetic pole 5 minutes, then directly pour out cell suspension.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN2011100797890A CN102719398A (en) | 2011-03-31 | 2011-03-31 | Novel efficient method for separating human T-Lymphocytes by immunomagnetic beads |
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| CN2011100797890A CN102719398A (en) | 2011-03-31 | 2011-03-31 | Novel efficient method for separating human T-Lymphocytes by immunomagnetic beads |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103305463A (en) * | 2013-06-05 | 2013-09-18 | 南昌大学 | Method for separating CD4<+> and CD25<+> lymphocytes in human peripheral blood by use of magnetic beads |
| CN103305464A (en) * | 2013-06-05 | 2013-09-18 | 南昌大学 | Method for directly separating CD<4+> and CD<8+> lymphocytes |
| CN103305462A (en) * | 2013-06-05 | 2013-09-18 | 南昌大学 | Method for enriching and separating human peripheral blood CD34+ and CD91+ lymphocytes |
| CN104046591A (en) * | 2014-06-24 | 2014-09-17 | 南昌大学 | Establishment of in-vitro memory B cell separating method |
| CN105651680A (en) * | 2016-02-04 | 2016-06-08 | 关节动力安达（天津）生物科技有限公司 | Method for identifying cell sorting efficiency of different immunomagnetic bead cell separators |
| CN106350485A (en) * | 2016-08-24 | 2017-01-25 | 杭州百凌生物科技有限公司 | Method for rapidly and efficiently separating individual antigen specific cell B |
| CN107435039A (en) * | 2017-06-13 | 2017-12-05 | 安徽安龙基因医学检验所有限公司 | A kind of preparation method and storage method of the immunomagnetic beads for sorting leucocyte |
| CN109679903A (en) * | 2019-01-16 | 2019-04-26 | 深圳咖荻生物科技有限公司 | Separation and purification method of T lymphocytes |
-
2011
- 2011-03-31 CN CN2011100797890A patent/CN102719398A/en active Pending
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103305463A (en) * | 2013-06-05 | 2013-09-18 | 南昌大学 | Method for separating CD4<+> and CD25<+> lymphocytes in human peripheral blood by use of magnetic beads |
| CN103305464A (en) * | 2013-06-05 | 2013-09-18 | 南昌大学 | Method for directly separating CD<4+> and CD<8+> lymphocytes |
| CN103305462A (en) * | 2013-06-05 | 2013-09-18 | 南昌大学 | Method for enriching and separating human peripheral blood CD34+ and CD91+ lymphocytes |
| CN103305462B (en) * | 2013-06-05 | 2015-05-20 | 南昌大学 | Method for enriching and separating human peripheral blood CD34+ and CD91+ lymphocytes |
| CN103305463B (en) * | 2013-06-05 | 2015-05-20 | 南昌大学 | Method for separating CD4<+> and CD25<+> lymphocytes in human peripheral blood by use of magnetic beads |
| CN104046591A (en) * | 2014-06-24 | 2014-09-17 | 南昌大学 | Establishment of in-vitro memory B cell separating method |
| CN104046591B (en) * | 2014-06-24 | 2016-08-24 | 南昌大学 | A kind of preparation method of in-vitro separation memory B cells |
| CN105651680A (en) * | 2016-02-04 | 2016-06-08 | 关节动力安达（天津）生物科技有限公司 | Method for identifying cell sorting efficiency of different immunomagnetic bead cell separators |
| CN106350485A (en) * | 2016-08-24 | 2017-01-25 | 杭州百凌生物科技有限公司 | Method for rapidly and efficiently separating individual antigen specific cell B |
| CN107435039A (en) * | 2017-06-13 | 2017-12-05 | 安徽安龙基因医学检验所有限公司 | A kind of preparation method and storage method of the immunomagnetic beads for sorting leucocyte |
| CN109679903A (en) * | 2019-01-16 | 2019-04-26 | 深圳咖荻生物科技有限公司 | Separation and purification method of T lymphocytes |
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Application publication date: 20121010 |