CN110396498A - The method of Activated in Vitro memory B cells plasmablast - Google Patents

The method of Activated in Vitro memory B cells plasmablast Download PDF

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CN110396498A
CN110396498A CN201810380061.3A CN201810380061A CN110396498A CN 110396498 A CN110396498 A CN 110396498A CN 201810380061 A CN201810380061 A CN 201810380061A CN 110396498 A CN110396498 A CN 110396498A
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cell
memory
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cpg
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杨希
葛良鹏
黄楠
郎巧利
余琳
何麒麟
吴梦
邹贤刚
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Chongqing Academy of Animal Sciences
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/52—CD40, CD40-ligand (CD154)
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/99—Coculture with; Conditioned medium produced by genetically modified cells

Abstract

The invention belongs to the culture of B cell or maintain technical field, more particularly to a kind of method of Activated in Vitro memory B cells plasmablast, the following steps are included: using the NIH-3T3/mCD40L cell of mitomycin C processing as feeder cells, the IMDM of 10%-20%FBS is as basic culture medium, using CpG, IL-2 and IL-21 as inducer, inducing memory B cell is converted into thick liquid cell and secretory antibody in vitro.This method can significantly improve activation efficiency, improve active mass；This method simple process and low cost.

Description

The method of Activated in Vitro memory B cells plasmablast
Technical field
The invention belongs to the culture of B cell or maintain technical field, and in particular to a kind of Activated in Vitro memory B cells at The method of thick liquid cell.
Background technique
After memory B cells are primary immune response, a kind of energy with memory function that B cell is broken up by antigenic activation Continue existing important immunocyte.When encountering same antigen again, memory B cells can Proliferation, Differentiation be rapidly that slurry is thin Born of the same parents generate the specific antibody of high-affinity, so that humoral immunity response, helps body to resist the invasion again of antigen. Memory B cells can play an important role in long-term existence body in antibody drug research.Currently, point of memory B cells From method relative maturity, but Activation In Vitro method is cumbersome, and at high cost, activity ratio is low, it is difficult to promote.Therefore, how efficiently in body It is outer to convert thick liquid cell as research hotspot for memory B cells.
2002, the discovery such as Bernasconi CpG and IL-15 collective effect can promote memory B cells and break up and rise in value (Maintenance of serological memory by polyclonal activation of human memory B cells,Bernasconi N L,et al.,Science,2002,298(5601):2199-2202).2004, Cortty etc. Memory B cells (Tracking human antigen-specific has been activated using CpG, PWM and staphylococcal protein A memory B cells:a sensitive and generalized ELISPOT system,Crotty S,et al., Journal of Immunological Methods,2004,286(1–2):111-122).2007, the discovery such as Ertesvag Increment (the Vitamin A potentiates CpG- of the CD27+ memory B cells of CpG mediation can be enhanced in vitamin A acid mediated memory B-cell proliferation and differentiation:involvement of early activation of p38MAPK,Ertesvag A,et al,Blood,2007,109(9):3865-72).2009, The discovery such as Pinna R848, IL-2 and CD40L, which surely turn cell, can activate memory B cells (Clonal dissection of the human memory B-cell repertoire following infection and vaccination,Pinna D,et al.,European Journal of Immunology,2009,39(5):1260–1270).2011, the benefits such as Wand It is thick liquid cell and successful expression antibody with the memory B cells activation of the Dendritic Cells of activation and induced t cell (Cooperation of dendritic cells with native lymphocyte populations to induce the generation of antigen-specific antibodies in vitro,Wand I,et al.,Journal of Biotechnology,2011,156(3):173-181)；Meanwhile grace that et al. utilizes IL-1 β, TNF-α, golden yellow Portugal Grape coccus Cowans strain cell, BAFF, IL-2, IL-10, IL-6 and IL-4 have activated gene traits B cell in vitro (CN102918395B).2014, Chen Tingtao etc. successfully induced note using CpG, IL-2, IL-10, B95-8 cells and supernatant The property recalled B cell is converted into thick liquid cell (CN104031880A).2016, Hu Ping etc. utilized IL-2I, L-21 and irradiation inactivated 3T3-CD40L cells in vitro inducing memory B cell activates plasmablast and secretory antibody (CN106222137A).These methods are all The defects of different degrees of has activated memory B cells, but there is complex process, at high cost.Wherein, in the method for Hu Ping etc. It is the most convenient and simple, but its activation efficiency is not high, active mass is lower.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of method of Activated in Vitro memory B cells plasmablast, it should The activation efficiency of method is high, and active mass is good, and simple process and low cost.
Unless otherwise indicated, percentage of the present invention is percent by volume.
To achieve the above object, the technical solution of the present invention is as follows:
The method of Activated in Vitro memory B cells plasmablast, comprising the following steps: the NIH- handled with mitomycin C 3T3/mCD40L cell is as feeder cells, and the IMDM of 10%-20%FBS is as basic culture medium, with CpG, IL-2 and IL- 21 are used as inducer, and inducing memory B cell is converted into thick liquid cell and secretory antibody in vitro.
The full name in English of the FBS is fetal bovine serum, and it is by plasma removing that Chinese translation, which is fetal calf serum, Fibrin and formed one kind it is light yellow clarification, without haemolysis, the slightly sticky thick liquid of foreign.
The full name in English of the DMEM is dulbecco ' s modified eagle medium, and Chinese translation is improvement Du Family name's Eagle's medium is a kind of culture medium containing various amino acid and glucose, is developed on the basis of MEM culture medium 's.Various composition dosage is increased compared with MEM.
The full name in English of the CpG is Class B CpG oligonucleotide, and Chinese translation is Type B CpG few nucleosides Acid.CpG is a kind of artificial synthesized oligonucleotides containing unmethylated CpG.CpG of the invention is public purchased from InvivoGen (Mouse specific) its sequence of ODN 1826 of department is " 5 '-tccatgacgttcctgacgtt-3 ' (20mer) ".
The full name in English of the IL-2 is interleukin-2, and Chinese translation is interleukin 2 also known as T cell growth The factor (T cell growth factor, abbreviation TCGF).
The full name in English of the IL-21 is interleukin-21, and Chinese translation is interleukin 21.
Inventors be surprised to learn that this method can significantly improve activation efficiency, active mass is improved.
Further, the content of FBS is 10% in the basal medium.
Further, described handled with mitomycin C refers to the mitomycin C that concentration is 10 μ g/mL at a temperature of 37 DEG C Handle 1-2h.
Further, described handled with mitomycin C refers to the mitomycin C that concentration is 10 μ g/mL at a temperature of 37 DEG C Handle 2h.
Further, the dosage of the IL-2 is that concentration 8-16ng/mL, the IL-21 dosage of IL-2 in culture medium is made to be to make The concentration of IL-21 is 50-100ng/mL in culture medium, and the dosage of CpG is to make 2.5 μ g/mL of CpG concentration in culture medium.
Further, the dosage of the IL-2 is that concentration 8ng/mL, the IL-21 dosage of IL-2 in culture medium is made to be to make to cultivate The concentration of IL-21 is 50ng/mL in base, and the dosage of CpG is the concentration 2.5ng/mL for making CpG in culture medium.
Further, described external evoked to specifically refer in 37 DEG C, 5%CO2Air in cultivate.
Further, it the described method comprises the following steps:
A. the preparation of feeder cells: by feeder cells NIH-3T3/mCD40L with the mitomycin C of 10 μ g/mL in 37 DEG C of temperature Degree is lower to handle 1-2h, then cleans cell with sterile PBS buffer, handles cell with the trypsin solution of 0.05%-0.25%, then Centrifugation removal precipitating, then with the DMEM of 10%-20%FBS resuspension cell, inoculated and cultured, for use；
B. it the preparation of splenic lymphocytes: is sterilized after killing immune mouse, then separating spleen is put into vessel, and uses scissors Spleen is shredded, PBS buffer solution is added and soaks spleen, is filtered after tissue block is milled, collects cell suspension；By cell suspension from Supernatant is abandoned after the heart, and erythrocyte cracked liquid is added in precipitating, is stood after mixing；Then FBS is added in cell suspension, after centrifugation Supernatant is abandoned, precipitating is resuspended with buffer；
C. the sorting of memory B cells: then the non-memory property B cell in removal mouse spleen memory B cells is collected And mark memory B cells；
D. the activation culture of memory B cells: preparing activation medium IMDM+10%FBS, and IL-2 be added wherein, IL-21 and CpG, so that the concentration of IL-2 is 8-32ng/ml in culture medium, the concentration of IL-21 is 50-100ng/ml, and CpG's is dense Degree is 2.5 μ g/ml, then by memory B cell in 37 DEG C, 5%CO2Air in cultivate.
The full name in English of the PBS is phosphate buffer saline, and Chinese translation is phosphate buffer.
This method can significantly improve activation efficiency, improve active mass.
This method simple process and low cost.
The beneficial effects of the present invention are:
This method can significantly improve activation efficiency, improve active mass.
This method simple process and low cost.
Specific embodiment
Illustrated embodiment is to preferably be illustrated to the contents of the present invention, but is not that the contents of the present invention only limit In illustrated embodiment.So those skilled in the art carry out nonessential change to embodiment according to foregoing invention content Into and adjustment, still fall within protection scope of the present invention.
The NIH-3T3 cell for the overexpression mouse CD40L that following NIH-3T3/mCD40L is constructed from this laboratory System, specific construction step are as follows: CD40L gene is synthesized according to the mRNA sequence of mouse CD40L in NCBI, is inserted into big expression vector Recombinant plasmid pcDNA3.1-CD40L is obtained in pcDNA3.1, pcDNA3.1-CD40L plasmid is transferred to by the method for recycling electricity to turn In NIH-3T3 cell (ATCC purchase), by G418 screen obtain stablize overexpression mouse CD40L cell line (referring to " Immunization against Endogenous Retroviral Tumor-associated Antigens ", MH Kershaw, et al., Cancer Research, 2001,61 (21): 7920-7924, publication date on November 30th, 2001)；
Mitomycin C is purchased from Sigma-Aldrich company；
The brand of FBS and DMRM is Gibico, is purchased from Thermo Fisher company；
The immune mouse of OVA is immunized normal C57/BL6 mouse for the OVA albumen (sigma company) of this laboratory purchase and obtains ；
(30 μm) of pre-separation filter Pre-Separation Filters are purchased from Miltenyi Biotec, and article No. is 130-041-407；
Memory B Cell Isolation Kit mouse kit from Mei Tian Ni Biotechnology Co., Ltd, Product batch number is 130-095-838.
Embodiment 1
The method of Activated in Vitro memory B cells plasmablast, specific steps are as follows:
A. the preparation of feeder cells: feeder cells NIH-3T3/mCD40L is with the mitomycin C of 10 μ g/mL in 37 DEG C of conditions Lower processing 2h, then cleaned cell 4 times with sterile 1 × PBS buffer solution, cell is handled with 0.05% trypsin solution, is centrifuged off After pancreatin, cell is resuspended with the DMEM containing 10%FBS, according to 5 × 104The density in a/hole is inoculated in 96 orifice plates, in 37 DEG C, 5%CO2Air in overnight incubation it is stand-by；
B. the preparation of splenic lymphocytes: choosing OVA and mouse be immunized, and puts to death immune mouse, 75% wine using cervical dislocation Smart solution impregnates 3-5min, opens left abdomen skin in super-clean bench, careful separation subcutaneous tissue and abdominal muscles expose spleen Dirty, separating spleen is put into vessel, is shredded spleen with scissors, and PBS buffer solution of the 0.5mL containing 2%FBS is added and soaks spleen, uses 2.5mL syringe piston gently mills tissue block, is then filtered with 30 μm of pre-separation filters, collects cell suspension, will receive The cell suspension collected is centrifuged 3min in the revolving speed of 1200 turns/min, abandons supernatant；3mL erythrocyte splitting is added in the precipitating of collection Liquid mixes gently, stands 3min；1mL FBS is drawn with pasteur pipet to be slowly added in cell suspension from bottom, in 1200 turns/ The revolving speed of min is centrifuged 3min, abandons supernatant, obtained precipitating is resuspended with buffer, is counted with haemocyte plate, 1.2 × 108 A/mL, total 3mL；
C. the sorting of memory B cells: Memory B Cell Isolation Kit mouse kit separating mouse is used Spleen memory B cells are specifically divided into two steps；(1) Memory B Cell the removal of non-memory property B cell: is added in the sample Biotin-Antibody Cocktail and Anti-Biotin MicroBeads, adds Anti-IgG1-APC and Anti- IgG2ab-APC antibody then uses LD column Magneto separate；(2) memory B cells are collected: between Anti-APC MicroBeads Label memory B cells are connect, using MS column Magneto separate, the cell for collecting separation is memory B cells；
D. the activation culture of memory B cells: preparing activation medium IMDM+10%FBS, and calculating needs medium body Product, is respectively set control group and experimental group, IL-2 and IL-21 is added in control group in the medium, and IL-2 concentration is in culture medium 8ng/mL, IL-21 concentration are 50ng/mL, will the obtained memory B cells of sorting according to 0 cells/well, 1 cells/well, 10 thin Born of the same parents/hole, 100 cells/wells, the density of 1000 cells/wells and 10000 cells/wells are inoculated with respectively in 96 well culture plates；Experimental group exists IL-2, IL-21 and CpG ODN 1826 is added in culture medium, IL-2 concentration is 8ng/mL in culture medium, and IL-21 concentration is 1826 concentration of 50ng/mL, CpG ODN is 2.5 μ g/ml, by the obtained memory B cells of sorting according to 0 cells/well, 1 cell/ Hole, 10 cells/wells, 100 cells/wells, the density of 1000 cells/wells and 10000 cells/wells are inoculated with respectively in 96 well culture plates. Control group and all cell plates of experimental group are in 37 DEG C, 5%CO2Air in cultivate two weeks, allow memory B cells activation to become anti- During which body secretory cell pays attention to observing cell state, collects supernatant and be used to detect in next step；
E. it tests: being coated with 96 orifice plates (100 μ L) with the OVA albumen in the hole 100ng/, 4 DEG C overnight, and board-washing machine is with 0.05% The PBS buffer solution board-washing of Tween (tween) 3 times；With the PBS for containing 1%BSA (bovine serum albumin(BSA)), 200 holes μ l/, 37 DEG C of closings Handle 2h, board-washing 3 times；Cell supernatant to be measured is diluted with volume ratio 1: 4, the volume of addition is 100 μ L；Room temperature acts on 1h, with The PBS board-washing of 0.05%Tween (tween) 3 times, then 100 μ L, 1: 1000 diluted HRP (horseradish mistake of volume ratio is added in every hole Oxide enzyme) label mouse IgG (immunoglobulin G)；100 hole μ l/ of OPD (o-phenylenediamine) developing solution is added, room temperature is protected from light Develop the color 20-30min, and 100 μ l 2.5mol/L sulfuric acid solution color development stoppings are added, and reads at OD450 (maximum absorption band 450nm) The value in each hole is taken, as a result as shown in Table 1 and Table 2, wherein table 1 is antibody level test result in control group, and table 2 is experimental group Middle antibody expression test result.
1 antibody expression test result (control group) of table
Cell number Hole 1 Hole 2 Hole 3 Hole 4 Hole 5 Hole 6 Hole 7 Hole 8
0/hole 0.075 0.085 0.092 0.073 0.081 0.087 0.083 0.079
1/hole 0.09 0.111 0.085 0.097 0.081 0.068 0.1 0.081
1/hole 0.141 0.303 0.181 0.24 0.167 0.143 0.207 0.206
1/hole 0.08 0.107 0.131 0.154 0.069 0.071 0.086 0.11
10/hole 0.083 0.101 0.117 0.082 0.105 0.067 0.106 0.109
100/hole 0.178 0.19 0.261 0.287 0.105 0.108 0.209 0.141
1000/hole 0.142 0.232 0.227 0.377 0.149 0.269 0.156 0.162
10000/hole 0.173 0.441 0.382 0.395 0.303 0.238 0.3 0.242
2 antibody expression test result (experimental group) of table
Cell number Hole 1 Hole 2 Hole 3 Hole 4 Hole 5 Hole 6 Hole 7 Hole 8
0/hole 0.087 0.068 0.091 0.086 0.075 0.093 0.079 0.077
1/hole 0.092 0.097 0.132 0.103 0.097 0.083 0.137 0.09
1/hole 0.153 0.326 0.203 0.285 0.229 0.181 0.237 0.259
1/hole 0.104 0.206 0.194 0.252 0.133 0.311 0.119 0.17
10/hole 0.089 0.449 0.155 0.415 0.134 0.196 0.12 0.346
100/hole 1.228 1.482 1.675 1.366 1.137 1.159 1.216 1.669
1000/hole 2.742 3.234 0.215 1.036 0.652 2.612 0.178 1.783
10000/hole 2.956 2.843 2.952 2.722 2.631 3.219 2.741 3.521
By Tables 1 and 2 it is found that compared with the control group, the method for experimental group can significantly improve in memory B cell supernatant Antibody expression.
Embodiment 2
The method of Activated in Vitro memory B cells plasmablast, specific steps are as follows:
A. the preparation of feeder cells: feeder cells NIH-3T3/mCD40L is with the mitomycin C of 10 μ g/mL in 37 DEG C of conditions Lower processing 2h, then cleaned cell 4 times with sterile 1 × PBS buffer solution, cell is handled with 0.05% trypsin solution, is centrifuged off After pancreatin, cell is resuspended with the DMEM containing 10%FBS, according to 5 × 104The density in a/hole is inoculated in 96 orifice plates, in 37 DEG C, 5%CO2Air in overnight incubation it is stand-by；
B. the preparation of splenic lymphocytes: choosing OVA and mouse be immunized, and puts to death immune mouse, 75% wine using cervical dislocation Smart solution impregnates 3-5min, opens left abdomen skin in super-clean bench, careful separation subcutaneous tissue and abdominal muscles expose spleen Dirty, separating spleen is put into vessel, is shredded spleen with scissors, and PBS buffer solution of the 0.5mL containing 2%FBS is added and soaks spleen, uses 2.5mL syringe piston gently mills tissue block, is then filtered with 30 μm of pre-separation filters, collects cell suspension, will receive The cell suspension collected is centrifuged 3min in the revolving speed of 1200 turns/min, abandons supernatant；3mL erythrocyte splitting is added in the precipitating of collection Liquid mixes gently, stands 3min；1mL FBS is drawn with pasteur pipet to be slowly added in cell suspension from bottom, in 1200 turns/ The revolving speed of min is centrifuged 3min, abandons supernatant, obtained precipitating is resuspended with buffer, is counted with haemocyte plate, 1.2 × 108 A/mL, total 3mL；
C. the sorting of memory B cells: Memory B Cell Isolation Kit mouse kit separating mouse is used Spleen memory B cells are specifically divided into two steps；(1) Memory B Cell the removal of non-memory property B cell: is added in the sample Biotin-Antibody Cocktail and Anti-Biotin MicroBeads, adds Anti-IgG1-APC and Anti- IgG2ab-APC antibody then uses LD column Magneto separate；(2) memory B cells are collected: between Anti-APC MicroBeads Label memory B cells are connect, using MS column Magneto separate, the cell for collecting separation is memory B cells；
D. the activation culture of memory B cells: preparing activation medium IMDM+10%FBS, and calculating needs medium body Product, and IL-2, IL-21 and CpG ODN 1826 is added wherein, IL-2 concentration is 8ng/mL, 32ng/mL, IL- in culture medium 21 concentration are 50ng/mL, 100ng/mL, and 1826 concentration of CpG ODN is 0 μ g/ml, 2.5 μ g/ml, carries out orthogonal test, will be divided Obtained memory B cells are selected to be inoculated in the version of 24 holes according to 50000 cells/wells, by all cell plates in 37 DEG C, 5%CO2's It is cultivated two weeks in air, memory B cells activation is allowed to become antibody secreting cell, during which pay attention to observing cell state, in collection Clear liquid for detecting in next step；
E. it tests: being coated with 96 orifice plates (100 μ L) with the OVA albumen in the hole 100ng/, 4 DEG C overnight, and board-washing machine is with 0.05% The PBS buffer solution board-washing of Tween (tween) 3 times；With the PBS for containing 1%BSA (bovine serum albumin(BSA)), 200 holes μ l/, 37 DEG C of closings Handle 2h, board-washing 3 times；Cell supernatant to be measured is diluted with volume ratio 1: 4, the volume of addition is 100 μ L；Room temperature acts on 1h, with The PBS board-washing of 0.05%Tween (tween) 3 times, then 100 μ L, 1: 1000 diluted HRP (horseradish mistake of volume ratio is added in every hole Oxide enzyme) label mouse IgG (immunoglobulin G)；1/ hole 100 μ of OPD (o-phenylenediamine) developing solution is added, room temperature is protected from light Develop the color 20-30min, and 100 μ l 2.5mol/L sulfuric acid solution color development stoppings are added, and reads at OD450 (maximum absorption band 450nm) The value in each hole is taken, the results are shown in Table 3.
Antibody expression test result under the conditions of 3 various concentration of table
Remarks :-indicate to be not added with.
In addition, it should be understood that although this specification is described in terms of embodiments, but not each embodiment is only wrapped Containing an independent technical solution, this description of the specification is merely for the sake of clarity, and those skilled in the art should It considers the specification as a whole, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the art The other embodiments being understood that.

Claims (8)

1. the method for Activated in Vitro memory B cells plasmablast, which comprises the following steps: at mitomycin C The NIH-3T3/mCD40L cell of reason as feeder cells, the IMDM of 10%-20% FBS as basic culture medium, with CpG, IL-2 and IL-21 is as inducer, and inducing memory B cell is converted into thick liquid cell in vitro.
2. the method according to claim 1, wherein the content of FBS is 10% in the basal medium.
3. method according to claim 1 or 2, which is characterized in that described handled with mitomycin C refers to that with concentration be 10 The mitomycin C of μ g/mL handles 1-2 h at a temperature of 37 DEG C.
4. according to the method described in claim 3, it is characterized in that, described handled with mitomycin C refers to that with concentration be 10 μ g/ The mitomycin C of mL handles 2 h at a temperature of 37 DEG C.
5. method according to claim 1,2,3 or 4, which is characterized in that the dosage of the IL-2 is to make IL- in culture medium 2 concentration is 8-16 ng/mL, and IL-21 dosage is to make the dosage of concentration the 50-100 ng/mL, CpG of IL-21 in culture medium To make 2.5 μ g/mL of CpG concentration in culture medium.
6. according to the method described in claim 5, it is characterized in that, the dosage of the IL-2 is to make the concentration of IL-2 in culture medium For 8 ng/mL, IL-21 dosage is that the dosage of concentration 50 ng/mL, CpG of IL-21 in culture medium is made to be to make CpG in culture medium Concentration be 2.5 ng/mL.
7. according to claim 1, method described in 2,3,4,5 or 6, which is characterized in that described external evoked to specifically refer in 37 ℃、5%CO2Air in cultivate.
8. method according to claim 1,2,3,4,5,6 or 7, which comprises the following steps:
A. the preparation of feeder cells: by feeder cells NIH-3T3/mCD40L with the mitomycin C of 10 μ g/mL in 37 DEG C of temperature Lower processing 1-2 h, then clean cell with sterile PBS buffer handles cell with the trypsin solution of 0.05%-0.25%, then from Heart removal precipitating, then cell, inoculated and cultured is resuspended with the DMEM of 10%-20% FBS；
B. the preparation of splenic lymphocytes: sterilizing after killing immune mouse, and then separating spleen is put into vessel, and with scissors by spleen It is dirty to shred, PBS buffer solution is added and soaks spleen, is filtered after tissue block is milled, collects cell suspension；After cell suspension is centrifuged Supernatant is abandoned, erythrocyte cracked liquid is added in precipitating, is stood after mixing；Then FBS is added in cell suspension, is abandoned after centrifugation Precipitating is resuspended clear liquid with buffer；
C. the sorting of memory B cells: then the non-memory property B cell in removal mouse spleen memory B cells is collected and is marked Remember memory B cells；
D. the activation culture of memory B cells: preparing activation medium IMDM+10%FBS, and IL-2 be added wherein, IL-21 And CpG, so that the concentration of IL-2 is 8-32 ng/mL in culture medium, the concentration of IL-21 is 50-100 ng/mL, the concentration of CpG For 2.5 μ g/ml, then by memory B cell in 37 DEG C, 5%CO2Air in cultivate.
CN201810380061.3A 2018-04-25 2018-04-25 The method of Activated in Vitro memory B cells plasmablast Pending CN110396498A (en)

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