CN105651680A - Method for identifying cell sorting efficiency of different immunomagnetic bead cell separators - Google Patents
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- 210000004698 lymphocyte Anatomy 0.000 claims abstract description 17
- 230000000694 effects Effects 0.000 claims abstract description 15
- 238000010606 normalization Methods 0.000 claims abstract description 6
- 239000006148 magnetic separator Substances 0.000 claims abstract description 5
- 238000012360 testing method Methods 0.000 claims description 26
- 210000004369 blood Anatomy 0.000 claims description 20
- 239000008280 blood Substances 0.000 claims description 17
- 239000000203 mixture Substances 0.000 claims description 12
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- 239000006228 supernatant Substances 0.000 claims description 12
- 239000004793 Polystyrene Substances 0.000 claims description 8
- 229920002223 polystyrene Polymers 0.000 claims description 8
- 238000001514 detection method Methods 0.000 claims description 6
- 230000000527 lymphocytic effect Effects 0.000 claims description 6
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 claims description 5
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 5
- 229960002897 heparin Drugs 0.000 claims description 5
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- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical compound O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 description 2
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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
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Abstract
The invention discloses a method for identifying the cell sorting efficiency of different immunomagnetic bead cell separators. Lymphocyte CD3 antibodies marked with immunomagnetic beads serve as indexes, the percentage content of CD3+ positive cells is detected and analyzed with a flow cytometry, the relative percentage is worked out with a normalization method, the separation effects of the magnetic separators with different intensities for sorting the blood lymphocyte of multiple persons are compared, and thus the purpose of selecting the separator with the optimal magnetic field for sorting various cells is achieved. Due to the fact that the whole blood lymphocyte is wide in source, the method of detecting CD3+ cells with the flow cytometry is easy and convenient to implement; CD3+ is sorted with the immunomagnetic beads, operation is easy, and the cell separating capabilities of the immunomagnetic bead cell separators with different magnetic field intensities can be conveniently and quickly identified. A quick, convenient and effective selecting method can be provided for the immunomagnetic bead separator for sorting cells.
Description
Technical field
The present invention relates to the qualification of immuno magnetic cell separation device, especially a kind of method utilizing flow cytomery CD3+ cell to identify different immuno magnetic cell separation device sorting cells efficiency.
Background technology
Immuno magnetic cell separation technology is a kind of new immunological technique combined by distinctive to the high degree of specificity of immunological response and magnetic bead magnetic responsiveness, it is combined with the specific monoclonal antibody being connected on magnetic bead by functional group based on antigen, form Ag-Ab-magnetic bead immune complex, this complex has higher magnetic responsiveness, displacement under the effect of external magnet magnetic force, make complex and other separating substances, and reach the purpose of separation, concentration, purification microorganism or specific antigenic substance. This technology is with a wide range of applications in cell separation, albumen, immunology and microbiologic inhibition tests etc.
The purity of immunomagnetic beads cell separation and yield rate are critically depend on immunomagnetic beads and magnetic field intensity. Magnetic bead is containing ferroso-ferric oxide superparamagnetism microsphere, its surface markers active group such as amino, carboxyl, sulfydryl etc., is reacted and antibodies by covalency or non-covalent associations, forms immune magnetic microsphere. The performance of high-quality microcarrier is: suitable, homogeneous magnetic response intensity; Less, homogeneous particle diameter; Stable uniform, Specific adsorption surface property. And the magnetic field intensity of external magnetic field should be mated with magnetic bead, to reach the separating effect of the best. Too little magnetic bead, too little magnetic field, coupling etc. that stability is inadequate make purpose cell yield not high, and too big magnetic bead and magnetic field can affect again cytoactive etc.
Immuno magnetic cell separation device in the market has two types: the separator needing pillar to separate and the magnetic separators of high-intensity magnetic field. Each own different pluses and minuses, the former is based on Mei Tian Ni company of Germany, and the latter becomes main flow because of simple and convenient convenience. The magnetic field intensity of Mei Tian Ni company magnet is typically in about 200mt, relies on plug-in detached dowel magnetic field intensity to be amplified, make diameter be 50nm bead attachment in magnetic post, by eluting thus reaching the effect separated;Cell can carry out follow-up experiment without the magnetic bead that dissociates, but the cell state sorted out is not good enough. The dominant company of outer magnet sorted product be Dial Stemcell the leading company such as BD and some domestic companies, magnetic bead is typically in 200nm-1.5um, and magnetic field is more than 400mt, and the cell purity of separation is high; And flow cytomery is compatible. Market also has a lot of company provide homemade magnet to separate cell, wherein patent applied for have tabular, magnetic pen, trace, knockdown magnetic separation device etc.
In view of immunological magnetic bead sorting technology generalizes, how to identify that the magnetic field intensity of commercial existing separator the best is a pendulum big problem in face of researcher with manufacturing required magnetic field fast and effectively.
Summary of the invention
The technical problem to be solved is to provide a kind of method identifying different immunomagnetic beads cell separator sorting cells efficiency, it can be determined that include the effect of the sorting cells of all types of immuno magnetic cell separation device.
In order to solve above-mentioned technical problem, the technical solution used in the present invention is: a kind of method identifying different immunomagnetic beads cell separator sorting cells efficiency, with indicate magnetic bead lymphocyte CD 3 antibody for index, CD3+ positive cell percentage composition is analyzed with flow cytomery, and calculate relative percentage with normalization method, the relatively magnetic separators sorting lymphocytic separating effect of many human bloods of varying strength, to reach the separator choosing required magnetic field to sort various cell.
Specifically, comprise the following steps:
(1) taking heparin anticoagulated whole blood adds in polystyrene tube, is initially charged the erythrocyte cracked liquid that CD3 cell key player on a team's test kit carries, and sequentially adds the sorting buffer in CD3 cell key player on a team's test kit and the magnetic particle sneaked out in advance in advance, incubated at room;
(2) add magnetic bead sorting medium constant volume, put in the magnet of cell separator to be measured and sort, abandoning supernatant, repeats step (2);
(3) use PBS re-suspended cell, namely obtain CD3+ cell in human lymphocyte;
(4) after negative control, non-sorting cells and sorting, cell is separately added into anti-CD3antibody, mix homogeneously, and lucifuge is hatched, it is centrifuged and abandons supernatant, cell volume by volume concentration is that 1% paraformaldehyde is fixed, 4 DEG C of lucifuges, uses stream type cell analyzer detection CD3 cell proportion;
(5) BDFACSDiVa software analysis T lymphocyte CD 3+ positive cell percentage composition is adopted, and adopt normalizing computational methods, calculate the percentage contents of CD3+ positive cell in different people blood, with the ability of relatively various magnets sorting different people blood lymphocytes, thus selecting the separator in required magnetic field for sorting cells.
Putting in described step (2) in the magnet of cell separator to be measured and sort, sorting adopts manual mode, and magnet is toppled over up and down 10s together with test tube, returns to stand up position and stands 5min.
Putting in described step (2) in the magnet of cell separator to be measured and sort, sorting adopts machinery rotation mode, magnet rotates 1000r2min together with test tube and stands 3min.
In described step (2), magnetic bead sorting medium is the 0.01mol/LPBS buffer containing 1mmol/LEDTA, 2%FBS, and percent is volume ratio.
Described CD3 cell key player on a team's test kit is CD3 cell key player on a team's test kit of stemcell company.
Repeating step (2) in described step (2) is 3 times.
Described stream type cell analyzer is BD_FACSCanto_II type stream type cell analyzer, and optical maser wavelength is 488nm, and transmitting wavelength is 525nm.
The invention has the beneficial effects as follows: Whole blood lymphocyte wide material sources; Flow cytomery CD3+ cellular processes is easy; Immunological magnetic bead sorting CD3+ is easily operated, and the immuno magnetic cell separation device that can conveniently identify different magnetic field intensity divides cellifugal ability. A kind of quick, convenient, effective system of selection can be provided for the Beads enrichment device of sorting cells.
Accompanying drawing explanation
Fig. 1 is just choosing anticoagulated whole blood CD3 lymphocyte, and Stemcell magnet (900mt), domestic magnet (900mt 500mt) are manually toppled over and rotation mode sorting streaming result up and down.
Detailed description of the invention
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail:
A kind of method identifying different immunomagnetic beads cell separator sorting cells efficiency of the present invention, with indicate magnetic bead lymphocyte CD 3 antibody for index, CD3+ positive cell percentage composition is analyzed with flow cytomery, and calculate relative percentage with normalization method, the relatively magnetic separators sorting lymphocytic separating effect of many human bloods of varying strength, to reach the separator choosing required magnetic field to sort various cell.
Specifically, comprise the following steps:
(1) taking heparin anticoagulated whole blood adds in polystyrene tube, is initially charged the erythrocyte cracked liquid that CD3 cell key player on a team's test kit carries, and sequentially adds the sorting buffer in CD3 cell key player on a team's test kit and the magnetic particle sneaked out in advance in advance, incubated at room;
(2) add magnetic bead sorting medium constant volume, put in the magnet of cell separator to be measured and sort, abandoning supernatant, repeats step (2);
(3) use PBS re-suspended cell, namely obtain CD3+ cell in human lymphocyte;
(4) after negative control, non-sorting cells and sorting, cell is separately added into anti-CD3antibody, mix homogeneously, and lucifuge is hatched, it is centrifuged and abandons supernatant, cell volume by volume concentration is that 1% paraformaldehyde is fixed, 4 DEG C of lucifuges, uses stream type cell analyzer detection CD3 cell proportion;
(5) BDFACSDiVa software analysis T lymphocyte CD 3+ positive cell percentage composition is adopted, and adopt normalizing computational methods, calculate the percentage contents of CD3+ positive cell in different people blood, with the ability of relatively various magnets sorting different people blood lymphocytes, thus selecting the separator in required magnetic field for sorting cells.
Putting in described step (2) in the magnet of cell separator to be measured and sort, sorting adopts manual mode, and magnet is toppled over up and down 10s together with test tube, returns to stand up position and stands 5min.
Putting in described step (2) in the magnet of cell separator to be measured and sort, sorting adopts machinery rotation mode, magnet rotates 1000r2min together with test tube and stands 3min.
In described step (2), magnetic bead sorting medium is the 0.01mol/LPBS buffer containing 1mmol/LEDTA, 2%FBS, and percent is volume ratio.
Described CD3 cell key player on a team's test kit is CD3 cell key player on a team's test kit of stemcell company.
Repeating step (2) in described step (2) is 3 times.
Described stream type cell analyzer is BD_FACSCanto_II type stream type cell analyzer, and optical maser wavelength is 488nm, and transmitting wavelength is 525nm.
Disclosure address how to verify and provide a kind of verification method quick, simple for different types of immuno magnetic cell separation device by the capability problems of different immuno magnetic cell separation device sorting cells; And the computational methods of a kind of normalizing are provided, overcome the shortcoming that its cell numerical value difference of the different people of flow cytomery is difficult to lateral comparison, it is possible to compare the detection lymphocytic difference of different people in different time.
Specifically, carry out according to the following steps:
1, experiment material and method:
1.1 experiment materials:
(1) source of blood: the heparin anti-coagulating of clinical patients or the normal person that haves a medical check-up.
(2) magnetic field and supporting test tube: stemcell company immuno magnetic cell separation device of Canada (900mt); Domestic different magnetic field intensity separator (900mt 500mt); Different magnet pipes is polystyrene tube.
(3) reagent and instrument: magnetic bead sorting is with buffer (the 0.01mol/LPBS buffer containing 1mmol/LEDTA, 2%FBS, with 0.22 ��m of membrane filtration, 4 DEG C of storages); CD3 cell key player on a team's test kit (HumanWholeBloodCD3PositiveSelectionKit, STEMCELL company of Canada, EasySep, article No. 18081); The mouse anti human CD3 monoclonal antibody (BD company, the U.S.) of flag F ITC; FACSCanto_II flow cytometer (BD company, the U.S.); Generic centrifuge (Xiang Yi company, Hunan China); Motor stirrer (JJ-1A/B digital display powerful motor agitator, White Tower Xin Bao instrument plant of Jintan City of China)
1.2 experimental techniques
(1) immunomagnetic beads (key player on a team's method) sorts people's lymph CD3+ cell: uses CD3+ cell just sorting test kit (StemCellTechnologies, Canada, catalog:#18081) and sub-elects initial CD3+T cell.
1) 1. anticoagulant heparin whole blood 0.5ml adds in the polystyrene tube of 14ml (magnet dedicated pipe)
2) erythrocyte cracked liquid (key player on a team's test kit with) of 1XEasySep, mixing is added in the ratio of 1:1.
3) adding the sorting buffer in key player on a team's test kit, mixing according still further to the ratio of 25 �� L/mL, room temperature stands 15min;
4) adding the magnetic particle (EasySepMAGNETICNANOPARTICLES, 200nm) (25uL/ pipe) sneaked out in advance in advance, blow and beat at least 5 times, it is ensured that fully mix, room temperature stands 10min.
5) addition magnetic bead sorting medium is settled to 5ml. and mixes piping and druming 2-3 time gently, and this polystyrene tube is put into (1. stemcell separator 900mt magnet in different immuno magnetic cell separation devices; 2. domestic separator 900mt magnet; 3. domestic separator 500mt magnet.
6) sorting mode: 1. manual mode: magnet is toppled over up and down 10s together with test tube, returns to stand up position and stands 5min (do not shake or get rid of and fall to being suspended on the drop of the mouth of pipe), abandoning supernatant; 2. rotation mode: magnet is rotated together with test tube 1000r2min and stands 3min, abandoning supernatant; At this moment the cell of magnetic marker is still at former pipe.
7) from magnet, take out the magnetic bead sorting medium that polystyrene tube adds 5ml, gently piping and druming 2-3 time, mix cell suspension, place into magnet sorting (repeating 5), then abandon supernatant (repeating 6).
8) repeating step 5-7, in magnet, 3 times altogether, 5min separation, then take out polystyrene tube, with PBS 1mL re-suspended cell, for the CD3+ positive cell after sorting.
(2) process of non-sorting cells: take 1 fluidic cell dedicated pipe, add patient whole blood 100 �� L, add 2mL1 �� red and split (BD Products), lysis at room temperature 10min, centrifugal 1500r/min, 5min, abandon supernatant, add PBS repeated washing once, rear addition PBS 1mL re-suspended cell (1 �� 107/ mL). (every solencyte number is 5 �� 10 to be divided into 2 pipes again6/ mL): the 1st pipe is not added with any antibody, as negative tube; 2nd pipe adds 5 �� L Mus anti-CD3antibodies, for CD3 marker determination pipe;
(3) flow cytomery:
1) non-sorting cells group more than, magnet sorting group cell place in fluidic cell dedicated pipe, centrifugal 1000r/min, 5min, abandon supernatant, often pipe adds 0.5mLPBS buffer re-suspended cell (5 �� 106/ mL), add 5 �� L Mus anti-CD3antibodies, softly blow and beat mixing, after room temperature lucifuge hatches 20-30min, the 0.01MPBS, the 1000rpm*5min that add 1mL are centrifugal, abandon supernatant.Fixing with 1% paraformaldehyde of 500 �� L, re-suspended cell is transferred in streaming pipe. 4 DEG C keep in Dark Place;
2) BD_FACSCanto_II type stream type cell analyzer detection CD3+ positive cell ratio (optical maser wavelength is 488nm, and transmitting wavelength is 525nm) is used. Collect and measure 10000, cell.
(4) streaming interpretation of result
1) percentage composition (%) of BDFACSDiVa software analysis T lymphocyte CD 3 is adopted
2) because the index coefficient of variation in everyone blood of FCM analysis is very big, it is impossible to statistics. At this, use normalization method, with unsorted result for reference value, utilize formula: (experimental result-reference value)/reference value �� 100%, obtain the percentage contents (%) of CD3+ positive cell with its difference, such that it is able to the different blood sample of patient CD3+ positive cell efficiency that the different Beads enrichment device of lateral comparison separates.
2, result
Show from table 1, table 2. result:
1) the lymphocytes in blood CD3 positive cell number of different people keeps just 63.22% substantially, after the sorting of immuno magnetic cell separation device, CD3 positive cell number significantly improves, compare with not sorting group, import Stemcell separator separating effect improves 41.65%, and domestic separator 900mt magnet separating effect improves 36.88%, and 500mt magnet separating effect improves 17.98%
2) in same separation, Stemcell separator (900mt), domestic separator (900mt and 500mt) rotation separation results be better than manually sorting mode result: 94.2%vs86.52%; 92.4%vs85.40%;
86.4%vs72.24%
Table 1 is just being chosen anticoagulated whole blood CD3 lymphocyte, Stemcell separator (900mt), domestic separator (900mt 500mt)
Manual and rotation mode sorts streaming result
Note: 1.-6. represent the blood samples of patients number of detection,
Table 2 adopts normalization method Computation immunity Beads enrichment device sorting people's lymphocytic effect of anticoagulated whole blood CD3
3, conclusion
From above flow cytomery CD3 positive cell as a result, it is possible to be concluded that
1) magnetism intensity of magnet is more big, and separating effect is more good; Determine that 900mt magnet is for the best experiment magnet;
2) separating effect of the immuno magnetic cell separation device cell of import Stemcell company is better than domestic separator;
3) effect that rotation mode separates CD3+ cell is utilized to be superior to the same manual effect of magnetic force magnet.
Immuno magnetic cell separation cell method is widely used in the sorting of various types of cells, for research or clinical treatment. For high efficient sorting cell, existing business-like separator is of all kinds, because point method such as immunomagnetic beads, coupling is different, each the magnetism intensity of design is different, it is exactly a problem that the magnet how selecting suitable strength magnetic field most efficiently separates cell, and the magnet especially for designed, designed is particularly important. The present invention is with the CD3+ of immunomagnetic beads key player on a team's Whole blood lymphocyte for index, applying flow cytometry technology, the immuno magnetic cell separation device sorting lymphocytic effect of human blood of fast verification varying strength, the immuno magnetic cell separation device sorting cells for choosing optimum magnetic field provides a new verification method.
Embodiment described above is merely to illustrate technological thought and the feature of the present invention, its object is to make those skilled in the art it will be appreciated that present disclosure implementing according to this, the scope of the claims of the present invention only can not be limited with the present embodiment, what namely all disclosed spirit was made changes on an equal basis or modifies, and still drops in the scope of the claims of the present invention.
Claims (8)
1. the method identifying different immunomagnetic beads cell separator sorting cells efficiency, it is characterized in that, with indicate magnetic bead lymphocyte CD 3 antibody for index, CD3+ positive cell percentage composition is analyzed with flow cytomery, and calculate relative percentage with normalization method, the relatively magnetic separators sorting lymphocytic separating effect of many human bloods of varying strength, to reach the separator choosing required magnetic field to sort various cell.
2. the method identifying different immunomagnetic beads cell separator sorting cells efficiency according to claim 1, it is characterised in that comprise the following steps:
(1) taking heparin anticoagulated whole blood adds in polystyrene tube, is initially charged the erythrocyte cracked liquid that CD3 cell key player on a team's test kit carries, and sequentially adds the sorting buffer in CD3 cell key player on a team's test kit and the magnetic particle sneaked out in advance in advance, incubated at room;
(2) add magnetic bead sorting medium constant volume, put in the magnet of cell separator to be measured and sort, abandoning supernatant, repeats step (2);
(3) use PBS re-suspended cell, namely obtain CD3+ cell in human lymphocyte;
(4) after negative control, non-sorting cells and sorting, cell is separately added into anti-CD3antibody, mix homogeneously, and lucifuge is hatched, it is centrifuged and abandons supernatant, cell volume by volume concentration is that 1% paraformaldehyde is fixed, 4 DEG C of lucifuges, uses stream type cell analyzer detection CD3 cell proportion;
(5) BDFACSDiVa software analysis T lymphocyte CD 3+ positive cell percentage composition is adopted, and adopt normalizing computational methods, calculate the percentage contents of CD3+ positive cell in different people blood, with the ability of relatively various magnets sorting different people blood lymphocytes, thus selecting the separator in required magnetic field for sorting cells.
3. the method identifying different immunomagnetic beads cell separator sorting cells efficiency according to claim 2, it is characterized in that, described step (2) is put in the magnet of cell separator to be measured and sort, sorting adopts manual mode, magnet is toppled over up and down together with test tube 10s, returns to stand up position and stand 5min.
4. the method identifying different immunomagnetic beads cell separator sorting cells efficiency according to claim 2, it is characterized in that, described step (2) is put in the magnet of cell separator to be measured and sort, sorting adopts machinery rotation mode, magnet rotates 1000r2min together with test tube and stands 3min.
5. the method identifying different immunomagnetic beads cell separator sorting cells efficiency according to claim 2, it is characterized in that, in described step (2), magnetic bead sorting medium is the 0.01mol/LPBS buffer containing 1mmol/LEDTA, 2%FBS, and percent is volume ratio.
6. the method identifying different immunomagnetic beads cell separator sorting cells efficiency according to claim 2, it is characterised in that described CD3 cell key player on a team's test kit is CD3 cell key player on a team's test kit of stemcell company.
7. the method identifying different immunomagnetic beads cell separator sorting cells efficiency according to claim 2, it is characterised in that repeating step (2) in described step (2) is 3 times.
8. the method identifying different immunomagnetic beads cell separator sorting cells efficiency according to claim 2, it is characterized in that, described stream type cell analyzer is BD_FACSCanto_II type stream type cell analyzer, and optical maser wavelength is 488nm, and transmitting wavelength is 525nm.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111621477A (en) * | 2020-05-25 | 2020-09-04 | 合源生物科技(天津)有限公司 | T cell sorting method |
| CN111757928A (en) * | 2017-12-01 | 2020-10-09 | 环球生命科技咨询美国有限责任公司 | Methods for cell enrichment and isolation |
| 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 |
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| CN111621477A (en) * | 2020-05-25 | 2020-09-04 | 合源生物科技(天津)有限公司 | T cell sorting method |
| CN111621477B (en) * | 2020-05-25 | 2021-06-22 | 合源生物科技(天津)有限公司 | T cell sorting method |
| 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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