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Adoptive Transfer of Memory B Cells
记忆B细胞的过继转移   

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参见作者原研究论文

本实验方案简略版
Nature Immunology
Jun 2014

Abstract

The adoptive transfer of antigen-specific B cells into mice that cannot recognize that specific antigen has two main advantages. The first is determining exactly when the B cells were transferred and exposed to antigen. The second is that all B cells that can bind that antigen are the ones that were transferred; no new antigen-specific B cells will emerge from the bone marrow. Thus all B cells that were exposed to the antigen and still alive after at least 4 weeks (8 weeks or more is ideal), are memory B cells.

Splenic B cells from B1-8 mice were prepared with an EasySep Mouse B Cell Enrichment Kit according to the manufacturer’s protocol. Single-cell suspensions were transferred intravenously into tail veins of recipient mice. Approximately 1 million NP+ B cells were transferred per mouse. Approximately 12-24 h after transfer, mice were immunized intra-peritoneally with 50 µg of NP-CGG precipitated in alum.

Materials and Reagents

  1. Mice
    Any donor mice can be used, as long as the donor and recipients have the same background strain (i.e. BALB/C into BALB/c or Bl/6 into Bl/6) to prevent rejection issues. We selected transgenic donor mice that had an increased frequency of B cells specific for our antigen of interest, NP. This way we could be certain of the number of B cells specific for our antigen and these would be easy to identify by flow cytometry and elispot. However, wild-type mice will also respond to NP, just at a lower frequency.
    1. B1.8+/-+/- BALB/c mice
      Note: B1.8 KI BALB/c mice were generated as described (Sonoda et al., 1997) and maintained on the Jκ KO strain (Chen et al., 1993) to enrich the frequency of λ+ NP-specific B cells. B1-8 KI +/+ Jκ KO -/- mice were crossed to BALB/c mice from The Jackson Laboratory (Bar Harbor, ME) to generate B1.8+/-+/- BALB/c mice, which were used for naïve controls and for transfers of NP+ B cells used to generate MBCs.
    2. AM14 Tg x Vκ8R KI BALB/c mice were generated as described (Shlomchik et al., 1993; Hannum et al., 1996; Prak and Weigert, 1995), which were used as recipient mice for primary immunization
      Note: All mice were maintained under specific pathogen-free conditions. The Yale Institutional Animal Care and Use Committee approved all animal experiments.

  2. Immunizations
    For generating memory B cells in a primary response, mice were immunized intra-peritoneally with 50 µg of 4-hydroxy-3-nitrophenyl acetyl (NP)-Chicken Gamma Globulin (CGG) precipitated in alum. The ratio of NP to CGG ranged between 26 and 33. All mice were immunized at 6-12 week of age

  3. Isolation of B cells from donor mice
    1. 2 pairs sterile scissor and forceps
    2. Sterile frosted slides
    3. Sterile Petri dishes
    4. Autoclaved Pasteur pipettes
    5. 70% ethanol
    6. Sterile ACK (RBC lysing buffer) (Lonza, catalog number: 10-548E )
    7. 100 µM filter (BD Biosciences, catalog number: 340615 )
    8. Ice
    9. Conical tubes (14 ml v-bottom) (BD Biosciences, Falcon®)
    10. Falcon 14 ml polystyrene round-bottom tubes (BD Biosciences, catalog number: 352057 )
    11. Trypan blue solution (0.4%) (Life Technologies, catalog number: 15250-061 )
    12. EasySep™ Mouse B Cell Enrichment Kit (STEMCELL Technologies, catalog number: 19754 ). Components of kit:
      1. EasySep™ (Negative Selection) Mouse B Enrichment Cocktail, 0.5 ml
      2. EasySep™ Biotin Selection Cocktail, 1 ml (store at 4 °C)
      3. EasySep™ Magnetic Particles, 1 ml (store at 4 °C; turn centrifuge on and cool to 4 °C)
      4. Normal Rat serum, 1 ml (store at -20 °C)
    13. RPMI-1640 with L-glutamine (Sigma-Aldrich, catalog number: R8758 )
    14. Fetal Calf Serum (GE Healthcare HyCloneTM)
    15. HEPES 1 M (Corning Incorporated, catalog number: 25-060-Cl )
    16. Streptomycin/penicillin, 10,000 U/ml (Life Technologies, Gibco®, Catalog number 15140-122 )
    17. 2-mercaptoethanol (Sigma-Aldrich, catalog number: M3128 )
    18. PBS without Ca2+/Mg2+ (Life Technologies, Gibco®, catalog number: 10010-023 )
    19. Ethylenediaminetetraacetic Acid (EDTA) 0.5 M Solution (pH 8) (Thermo Fisher Scientific, catalog number: 25783 )
    20. 2.5% Anticoagulant citrate dextrose solution [ACD(A)] (Polymed, catalog number: 7300 )
      ACDA was from the blood bank (http://www.polymedicure.com/?wpcproduct=acd-bag). Each 100 ml of ACD solution-A contains 2.2 g sodium citrate, 0.73 g citric acid, 2.45 g dextrose and 100 ml water.
    21. NP-binding reagents: NP-allophycocyanin (APC) (Shlomchik lab)
    22. Anti-CD4 (GK1.5) (Shlomchik lab)
    23. anti-Fc gamma RIII/II (2.4G2) (Shlomchik lab)
    24. anti-CD19 (1D3.2) (Shlomchik lab)
    25. 27 G needle, 1 ml syringe
    26. Ethidium Monoazide (EMA) 2 mg/ml (Molecular Probes)
    27. Complete media (see Recipes)
    28. EasySep media (see Recipes)
    29. Transfer buffer (see Recipes)
    30. Staining Media (see Recipes)

Equipment

  1. Sterile hood
  2. Refrigerated table top centrifuge
  3. Hemocytometer
  4. “EasySep” magnet (max vol 8 ml; min vol 250 µl) (STEMCELL Technologies, catalog number: 18001 )

Procedure

  1. Isolation of B cells
    1. Set the centrifuge temperature to 4 °C.
    2. Put one petri dish per spleen on top of ice in an ice bucket with 5 ml of complete media per dish.
    3. Euthanize mice.
    4. Take dead mice to sterile hood to dissect.
    5. Remove spleens. Place the spleen in the petri dish of complete media on ice.
    6. Grind the spleen between the frosted surfaces of the slides until the mixture is fairly uniform. (Alternatives included crushing spleens using the tip of a syringe or using other methods). Rinse the slides with complete media and transfer the remaining liquid through a filter into a 15 ml conical tube. Keep on ice while collecting other spleens.
    7. Centrifuge the cells at 4 °C, 400 RCF, for 10 min.
      Note: This can vary between 8-15 min, depending on the centrifuge, available time, and concern for loss of cells.
    8. Remove tubes from centrifuge. Decant into a container with one swift motion (in the hood, to retain sterility). Resuspend pellet in remaining media after decanting by tapping.
    9. Add ACK (4 ml per spleen) to lyse the red blood cells. Incubate at RT for 4 min. During this incubation, remove connective tissue and membranes of lysed cells, which clump together and look like white filaments or “ghosts”, using a Pasteur pipette.
    10. Fill the conical tube to the top with complete media, invert to mix and centrifuge again at 4 °C, 400 RCF for 10 min.
    11. Vacuum up liquid (under sterile conditions) and resuspend the pellet in remaining media after decanting by tapping. Combine all spleens (keep genders separate).
    12. Count each sample in a hemocytometer. Collect 10 µl aliquot, make a dilution with Trypan blue 1:10 in PBS. Calculate volume needed for 100 x 106 cells/ml.
      (live cells) (dilution) (104) = cells/ml; (cells/ml) (volume) = total cells
      [Total cells]/[100x106 cells/ml] = volume needed for 108 cells/ml
    13. Resuspend to the correct volume in 95% EasySep medium and 5% rat serum at 108 cells/ml.

  2. EasySep B cell enrichment
    Note: Follow manufacturer’s indications, which can change.
    For processing 500 μl-8.0 ml of sample (< 8.0 x 108 cells)
    1. Save an aliquot of cells pre-depletion for FACS.
    2. Prepare cell suspension at 1 x 108 cells/ml in medium with 5% normal rat serum. Place cells in a 14 ml (17 x 100 mm) polystyrene tube.
    3. Add Negative Selection Mouse B cell Enrichment Cocktail at 50 μl/ml. Mix well and incubate on ice for 15 min.
    4. Add Biotin Selection Cocktail at 100 μl/ml. Mix well and incubate on ice for 15 min.
    5. Mix Magnetic Particles to ensure that they are in a uniform suspension by pipetting vigorously 5 times or vortexing quickly. Add the Magnetic Particles at 100 μl/ml. Mix well and incubate on ice for 5 min.
    6. Bring the cell suspension to a total volume of 5 ml (for < 4 x 108 cells) or 10 ml (for 4-8.5 x 108 cells) by adding medium without rat serum. Mix the cells in the tube by pipetting gently 2-3 times.
    7. Place the tube (without cap) into the magnet. Set aside for 5 min at room temperature.
    8. Pick up the magnet and in one continuous motion invert the magnet and tube, pouring off the desired fraction into a new 14 ml tube. Leave the magnet and tube in inverted position for 2-3 sec, and then return to the upright position. Do not shake or blot off any drops that may remain hanging from the mouth of the tube.
    9. Count cells using a hemocytometer.
    10. Check purity and antigen specific cell percentage by flow cytometry.
      1. Save at least 2 x 106 cells for staining.
      2. Place in 96-well plate for staining.
      3. Centrifuge at 280 RCF, 4 °C for 4 min.
      4. Prepare antibody cocktail assuming 50 μl for each sample, antibody mix: NP-APC, CD19-Pacific Blue, CD4-FITC.
      5. Decant residual volume in sink with a quick inversion.
      6. Resuspend pelleted cells in 50 μl of staining media.
      7. Add 50 μl of antibody cocktail to each well.
      8. Mix well with pipette up and down.
      9. Incubate for 20 min on ice, covered with aluminum foil.
      10. Add 100 μl of staining media.
      11. Centrifuge at 280 RCF, 4 °C for 4 min.
      12. Decant residual volume in sink with a quick inversion.
      13. Resuspend pelleted cells in ~175 μl of staining media.
      14. Centrifuge at 280 RCF, 4 °C for 4 min.
      15. Decant residual volume in sink with a quick inversion.
      16. Resuspend pellet in 175 μl of PBS.
      17. Transfer samples from 96-well plate into test tubes immediately before going to the flow cytometry facility.
      18. Add 0.02 μl of EMA per sample right before running on flow cytometer.

  3. Mouse Injection
    Determine number of cells needed. We would transfer 1 million antigen-specific cells per mouse. We would determine the percentage of antigen-specific B cells in a sample by flow cytometry and then transfer the total number of cells accordingly. We also determined the percentage purity of B cells by flow cytometry. The purity of B cells was typically 90%.
    Inject 1 million NP+ B viable cells suspended in transfer buffer per mouse intravenously in 0.2 ml volume.

Representative data



Figure 1. Flow cytometry of splenic cells from AM14-Tg x Vκ8R-KI recipient mice given NP-specific B cells and immunized with NP-CGG in alum, assessed 8 weeks later. Number adjacent to outlined area indicates percent CD19+NP+ antigen-specific B cells among live cells. (from Zuccarino-Catania et al., 2014)

Figure 2. Flow cytometry of splenic B cells from AM14-Tg x Vκ8R-KI mice immunized with NP-CGG in alum without transfer of NP-specific B cells, assessed 8 weeks later as in Figure 1. (from Zuccarino-Catania et al., 2014)

Notes

  1. Usually mice are dissected in a sterile hood, so that splenic B cells remain sterile after harvesting. Cells should be kept cold at all times to minimize death and activation prior to transfer (either on ice or in 4 °C fridge).
  2. Another option, instead of B cell enrichment, is to do a complement depletion of T cells. This is a bit cheaper and could yield similar levels of purity. We decided to use the EasySep method from the start and got good results, so we continued with this method. It is important to use a method of enriching B cells, without their activation. We also wanted to avoid transferring T cells from our donor mice, to avoid any rejection issues (our recipients had intact naïve T cells that would help the generation of memory B cells just as well as donor T cells).

Recipes

  1. Complete media
    443.5 ml RPMI-1640 w/L-glutamine (or add 5 ml L-glu to 500 ml RPMI)
    50 ml fetal calf serum
    5 ml HEPES (10 mM)
    1 ml streptomycin/penicillin
    0.5 ml 2-mercaptoethanol (50 mM)
    Combine ingredients and filter
    Keep in 4 °C fridge until needed
  2. EasySep media
    244.5 ml 1x PBS without Ca2+/Mg2+
    0.5 ml 0.5 M stock EDTA (final is 1 mM)
    5 ml 2% calf serum
  3. Transfer buffer
    50 ml 1x PBS without Ca2+/Mg2+, sterile filtered
    0.5 ml 10 mM HEPES
    0.25 ml streptomycin/penicillin
    1.25 ml 2.5% ACDA
  4. Staining media
    1 L 1x PBS without Ca2+/Mg2+
    30 ml fetal calf serum (3% final concentration)
    2.5 ml NaN3 stock (0.04%)

Acknowledgments

This protocol was developed or modified in Dr. Mark Shlomchik’s laboratory at Yale University. Supported by the National Institutes of Health (R01-AI46303 to M.J.S) and NSF Graduate Research fellowships (G.V.Z.-C.). This protocol was adapted from Tomayko et al. (2010) and Sweet et al. (2013).

References

  1. Chen, J., Trounstine, M., Kurahara, C., Young, F., Kuo, C., Xu, Y., Loring, J., Alt, F. and Huszar, D. (1993). B cell development in mice that lack one or both immunoglobulin kappa light chain genes. EMBO J 12(3): 821.
  2. Hannum, L. G., Ni, D., Haberman, A. M., Weigert, M. G. and Shlomchik, M. J. (1996). A disease-related rheumatoid factor autoantibody is not tolerized in a normal mouse: implications for the origins of autoantibodies in autoimmune disease. J Exp Med 184(4): 1269-1278.
  3. Prak, E. L. and Weigert, M. (1995). Light chain replacement: a new model for antibody gene rearrangement. J Exp Med 182(2): 541-548.
  4. Shlomchik, M. J., Zharhary, D., Saunders, T., Camper, S. A. and Weigert, M. G. (1993). A rheumatoid factor transgenic mouse model of autoantibody regulation. Int Immunol 5(10): 1329-1341.
  5. Sonoda, E., Pewzner-Jung, Y., Schwers, S., Taki, S., Jung, S., Eilat, D. and Rajewsky, K. (1997). B cell development under the condition of allelic inclusion. Immunity 6(3): 225-233.
  6. Sweet, R. A., Cullen, J. L. and Shlomchik, M. J. (2013). Rheumatoid factor B cell memory leads to rapid, switched antibody-forming cell responses. J Immunol 190(5): 1974-1981.
  7. Tomayko, M. M., Steinel, N. C., Anderson, S. M. and Shlomchik, M. J. (2010). Cutting edge: Hierarchy of maturity of murine memory B cell subsets. J Immunol 185(12): 7146-7150.
  8. Zuccarino-Catania, G. V., Sadanand, S., Weisel, F. J., Tomayko, M. M., Meng, H., Kleinstein, S. H., Good-Jacobson, K. L. and Shlomchik, M. J. (2014). CD80 and PD-L2 define functionally distinct memory B cell subsets that are independent of antibody isotype. Nat Immunol 15(7): 631-637.

简介

将抗原特异性B细胞过继转移到不能识别该特异性抗原的小鼠中具有两个主要优点。 第一种是确切地确定B细胞何时转移并暴露于抗原。 第二是可以结合该抗原的所有B细胞是转移的; 从骨髓中不会出现新的抗原特异性B细胞。 因此,暴露于抗原并在至少4周(8周或更长时间是理想的)后仍存活的所有B细胞是记忆B细胞。
根据制造商的方案用EasySep小鼠B细胞富集试剂盒制备来自B1-8小鼠的脾B细胞。 将单细胞悬浮液静脉内转移到受体小鼠的尾静脉中。 每只小鼠转移约100万个NP + B细胞。 转移后约12-24小时,用在明矾中沉淀的50μgNP-CGG腹膜内免疫小鼠。

材料和试剂

  1. 小鼠
    可以使用任何供体小鼠,只要供体和受体具有相同的背景菌株(即BALB/C进入BALB/c或B1/6进入B1/6)以防止排斥问题。我们选择具有对我们的感兴趣的抗原NP特异的B细胞频率增加的转基因供体小鼠。这样我们可以确定对我们的抗原特异的B细胞的数目,并且这些将容易通过流式细胞术和elispot鉴定。然而,野生型小鼠也将对NP响应,只是以较低的频率
    1. B1.8 +/- +/- BALB/c小鼠
      注意:B1.8KI BALB/c小鼠 (Sonoda等人,1997)产生并保持在JκKO上  菌株(Chen等,1993)以富集λ + NP-特异性B的频率 细胞。将B1-8KI +/+ JκKO /- 小鼠与BALB/c小鼠 Jackson Laboratory(Bar Harbor,ME)产生B1.8 +/- Jκ +/- BALB/c 小鼠,其用于初始对照和NP + 的转移 细胞用于产生MBC。

      材料和试剂

      1. 小鼠
        可以使用任何供体小鼠,只要供体和受体具有相同的背景菌株(即BALB/C进入BALB/c或B1/6进入B1/6)以防止排斥问题。我们选择具有对我们的感兴趣的抗原NP特异的B细胞频率增加的转基因供体小鼠。这样我们可以确定对我们的抗原特异的B细胞的数目,并且这些将容易通过流式细胞术和elispot鉴定。然而,野生型小鼠也将对NP响应,只是以较低的频率
        1. B1.8 +/- +/- BALB/c小鼠
          注意:B1.8KI BALB/c小鼠 (Sonoda等人,1997)产生并保持在JκKO上  菌株(Chen等,1993)以富集λ + NP-特异性B的频率 细胞。将B1-8KI +/+ JκKO /- 小鼠与BALB/c小鼠 Jackson Laboratory(Bar Harbor,ME)产生B1.8 +/- Jκ +/- BALB/c 小鼠,其用于初始对照和NP + 的转移 细胞用于产生MBC。
          ...

          材料和试剂

          1. 小鼠
            可以使用任何供体小鼠,只要供体和受体具有相同的背景菌株(即BALB/C进入BALB/c或B1/6进入B1/6)以防止排斥问题。我们选择具有对我们的感兴趣的抗原NP特异的B细胞频率增加的转基因供体小鼠。这样我们可以确定对我们的抗原特异的B细胞的数目,并且这些将容易通过流式细胞术和elispot鉴定。然而,野生型小鼠也将对NP响应,只是以较低的频率
            1. B1.8 +/- +/- BALB/c小鼠
              注意:B1.8KI BALB/c小鼠 (Sonoda等人,1997)产生并保持在JκKO上  菌株(Chen等,1993)以富集λ + NP-特异性B的频率 细胞。将B1-8KI +/+ JκKO /- 小鼠与BALB/c小鼠 Jackson Laboratory(Bar Harbor,ME)产生B1.8 +/- Jκ +/- BALB/c 小鼠,其用于初始对照和NP + 的转移 细胞用于产生MBC。
              ......

              材料和试剂

              1. 小鼠
                可以使用任何供体小鼠,只要供体和受体具有相同的背景菌株(即BALB/C进入BALB/c或B1/6进入B1/6)以防止排斥问题。我们选择具有对我们的感兴趣的抗原NP特异的B细胞频率增加的转基因供体小鼠。这样我们可以确定对我们的抗原特异的B细胞的数目,并且这些将容易通过流式细胞术和elispot鉴定。然而,野生型小鼠也将对NP响应,只是以较低的频率
                1. B1.8 +/- +/- BALB/c小鼠
                  注意:B1.8KI BALB/c小鼠 (Sonoda等人,1997)产生并保持在JκKO上  菌株(Chen等,1993)以富集λ + NP-特异性B的频率 细胞。将B1-8KI +/+ JκKO /- 小鼠与BALB/c小鼠 Jackson Laboratory(Bar Harbor,ME)产生B1.8 +/- Jκ +/- BALB/c 小鼠,其用于初始对照和NP + 的转移 细胞用于产生MBC。
                  .........
                  1. EasySep™(阴性选择)小鼠B浓缩鸡尾酒,0.5 ml
                  2. EasySep™生物素选择鸡尾酒,1 ml(4°C存储)
                  3. EasySep™磁性颗粒,1 ml(存储在4°C;离心机开启,冷却至4°C)
                  4. 正常大鼠血清,1ml(-20℃保存)
                2. RPMI-1640(含有L-谷氨酰胺)(Sigma-Aldrich,目录号:R8758)
                3. 胎牛血清(GE Healthcare HyClone TM
                4. HEPES 1M(Corning Incorporated,目录号:25-060-Cl)
                5. 链霉素/青霉素,10,000U/ml(Life Technologies,Gibco ,目录号15140-122)
                6. 2-巯基乙醇(Sigma-Aldrich,目录号:M3128)
                7. PBS(不含Ca 2+ 2 + /Mg sup 2 + (Life Technologies,Gibco ,目录号:10010-023)
                8. 乙二胺四乙酸(EDTA)0.5M溶液(pH 8)(Thermo Fisher Scientific,目录号:25783)
                9. 2.5%抗凝血柠檬酸葡萄糖溶液[ACD(A)](Polymed,目录号:7300)
                  ACDA来自血库 ( http://www.polymedicure.com/?wpcproduct=acd-bag ) 。 每100 ml ACD 溶液-A含有2.2g柠檬酸钠,0.73g柠檬酸,2.45g 葡萄糖和100ml水。
                10. NP结合试剂:NP-别藻蓝蛋白(APC)(Shlomchik lab)
                11. 抗CD4(GK1.5)(Shlomchik实验室)
                12. 抗-FcγRIII/II(2.4G2)(Shlomchik lab)
                13. 抗CD19(1D3.2)(Shlomchik lab)
                14. 27针,1毫升注射器
                15. 单乙酰胺(EMA)2mg/ml(Molecular Probes)
                16. 完成媒体(见配方)
                17. EasySep介质(参见配方)
                18. 传输缓冲区(请参阅配方)
                19. 染色介质(见配方)

              设备

              1. 无菌罩
              2. 冷冻台式离心机
              3. 血细胞计数器
              4. "EasySep"磁体(最大体积8ml;最小体积250μl)(STEMCELL Technologies,目录号:18001)

              程序

              1. B细胞的分离
                1. 将离心机温度设置为4°C
                2. 每个脾脏一个培养皿在冰上的冰桶,每个培养皿5毫升完全培养基。
                3. 安乐死小鼠。
                4. 取死的老鼠无菌罩解剖。
                5. 去除脾脏。 将脾脏置于冰上完整培养基的培养皿中。
                6. 研磨幻灯片的磨砂表面之间的脾脏直到 混合物相当均匀。 (替代品包括使用破碎脾脏   注射器的尖端或使用其他方法)。 冲洗幻灯片 完全介质并将剩余的液体通过过滤器转移到a   15 ml锥形管。 在收集其他脾脏时保持冰上。
                7. 在4℃,400RCF离心细胞10分钟。
                  注意:这可以在8-15分钟之间变化,这取决于离心机,可用时间以及对细胞丢失的担心。
                8. 从离心机中取出管。 滗入一个容器与一个swift 运动(在罩子里,保持无菌)。 将剩余的沉淀重悬   通过轻敲滗析后的介质。
                9. 添加ACK(每个脾4ml) 溶解红细胞。 在室温下孵育4分钟。 在此期间 孵育,去除结缔组织和裂解细胞的膜,   聚集在一起,看起来像白色的细丝或"鬼魂",使用 巴斯德吸管。
                10. 使用完全培养基将锥形管填充至顶部,倒置混合并在4℃,400RCF下再次离心10分钟。
                11. 真空吸干液体(在无菌条件下),并重悬沉淀   通过轻敲滗析后的剩余介质。 结合所有脾脏(保持 性别分开)。
                12. 在血细胞计数器中计数每个样品。 搜集 10μl等分试样,用PBS中的台盼蓝1:10稀释。 计算 100×10 6细胞/ml所需的体积 (活细胞)(稀释)(10 4次)=细胞/ml; (细胞/ml)(体积)=总细胞
                  [总细胞]/[100×10 6细胞/ml] = 10 8个细胞/ml所需的体积
                13. 在95%EasySep培养基和5%大鼠血清中以10 8个细胞/ml重悬至正确的体积。

              2. EasySep B细胞富集
                注意:按照制造商的说明进行更改。
                对于处理500μl-8.0ml样品(<8.0×10 8个细胞)
                1. 保存用于FACS的细胞预去除的等分试样
                2. 准备细胞 在具有5%正常大鼠血清的培养基中以1×10 8个细胞/ml悬浮。 地点   细胞在14ml(17×100mm)聚苯乙烯管中。
                3. 添加负选择鼠标B细胞浓缩鸡尾酒以50μl/ml。 充分混合并在冰上孵育15分钟
                4. 添加生物素选择鸡尾酒在100微升/毫升。 充分混合并在冰上孵育15分钟。
                5. 混合磁性颗粒以确保它们处于均匀悬浮液中 通过剧烈移液5次或快速涡旋。 添加磁性 颗粒在100μl/ml。 充分混合并在冰上孵育5分钟
                6. 将细胞悬浮液加入总体积为5ml(对于<4×10 8个细胞)或10ml(对于4-8.5×10 8个细胞),通过加入 培养基无大鼠 血清。 轻轻吸取管中的细胞混合2-3次。
                7. 将管(无盖)放入磁铁。 在室温下放置5分钟。
                8. 拿起磁铁,在一个连续的运动反转磁铁和 管,将所需级分倒入新的14ml管中。 离开 磁铁和管在倒置位置2-3秒,然后返回   直立姿势。 不要摇晃或吸掉可能残留的任何液滴 挂在管口上。
                9. 使用血细胞计数器计数细胞
                10. 通过流式细胞术检查纯度和抗原特异性细胞百分比
                  1. 保存至少2 x 10 6 个细胞用于染色
                  2. 置于96孔板中进行染色
                  3. 在280RCF,4℃离心4分钟
                  4. 准备抗体混合物,假设每个样品50μl,抗体混合物:NP-APC,CD19-太平洋蓝,CD4-FITC。
                  5. 通过快速反转来确定水槽中的残余体积。
                  6. 将沉淀的细胞重悬于50μl染色培养基中
                  7. 向每个孔中加入50μl抗体混合液。
                  8. 用移液管上下混匀。
                  9. 在冰上孵育20分钟,用铝箔覆盖
                  10. 加入100μl染色培养基。
                  11. 在280RCF,4℃离心4分钟
                  12. 通过快速反演确定水槽中的残余体积。
                  13. 将沉淀的细胞重悬于〜175μl染色培养基中
                  14. 在280RCF,4℃离心4分钟
                  15. 通过快速反转来确定水槽中的残余体积。
                  16. 重悬在175μlPBS中的沉淀。
                  17. 在进入流式细胞仪设施之前,立即将样品从96孔板转移到试管中
                  18. 在流式细胞仪上运行前,每个样品加入0.02μl的EMA。

              3. 鼠标注入
                确定所需的单元数。 我们将转移每个小鼠100万抗原特异性细胞。 我们将通过流式细胞术确定样品中抗原特异性B细胞的百分比,然后相应地转移细胞的总数。 我们还通过流式细胞术测定了B细胞的纯度百分比。 B细胞的纯度通常为90% 以0.2ml体积静脉内注射悬浮在转移缓冲液中的每只小鼠的100万个NP + B活细胞。

              代表数据



              图1.在给予NP特异性B细胞并在明矾中用NP-CGG免疫的AM14-Tg xVκ8R-KI受体小鼠脾细胞的流式细胞术,8周后评估。与轮廓区域相邻的数目表示活细胞中CD19 + NP +抗原特异性B细胞的百分比。 (来自Zuccarino-Catania等人,2014)

              图2.在没有转移NP特异性B细胞的明矾中用NP-CGG免疫的AM14-Tg xVκ8R-KI小鼠的脾脏B细胞的流式细胞术,如图1所示在8周后评估。(来自Zuccarino-卡塔尼亚等,2014)

              笔记


              1. 通常在无菌罩中解剖小鼠,使脾细胞 收获后保持无菌。 细胞应保持冷的任何时候 以最小化转移前的死亡和活化(在冰上或在4   °C冰箱)。
              2. 另一个选择,而不是B细胞富集,是做补充 消耗T细胞。 这有点便宜,可能产生类似 纯度水平。 我们决定从一开始就使用EasySep方法 并得到好的结果,所以我们继续这种方法。 这很重要 使用富集B细胞而不激活B细胞的方法。 我们也 想避免转移T细胞从我们的捐助小鼠,以避免任何 拒绝问题(我们的收件人完整的初始T细胞,会 有助于记忆B细胞的产生以及供体T细胞)。


              食谱

              1. 填写媒体
                443.5ml RPMI-1640 w/L-谷氨酰胺(或向500ml RPMI中加入5ml L-葡萄糖)
                50ml胎牛血清
                5ml HEPES(10mM) 1 ml链霉素/青霉素
                0.5ml 2-巯基乙醇(50mM) 组合原料和过滤器
                保存在4°C冰箱直到需要
              2. EasySep媒体
                244.5ml不含Ca 2+的1x PBS + /Mg 2 +
                0.5ml 0.5M储备EDTA(最终为1mM) 5ml 2%小牛血清
              3. 传输缓冲区
                50ml无1×PBS的PBS +无菌过滤的 2 + /Mg 0.5 ml 10 mM HEPES
                0.25ml链霉素/青霉素
                1.25ml 2.5%ACDA
              4. 染色介质
                1 L 1x PBS,不含Ca 2+ 2//Mg 2 +
                30ml胎牛血清(3%终浓度) 2.5ml NaN 3+原液(0.04%)

              致谢

              该协议在耶鲁大学的Mark Shlomchik实验室中开发或修改。由国家卫生研究院(R01-AI46303至M.J.S)和NSF研究生研究奖学金(G.V.Z.-C.)支持。该协议改编自Tomayko等人(2010)和Sweet等人(2013)。

              参考文献

              1. Chen,J.,Trounstine,M.,Kurahara,C.,Young,F.,Kuo,C.,Xu,Y.,Loring,J.,Alt,F.and Huszar,D。 缺乏一个或两个免疫球蛋白κ轻链基因的小鼠中的B细胞发育。 a> EMBO J 12(3):821.
              2. Hannum,L.G.,Ni,D.,Haberman,A.M.,Weigert,M.G.and Shlomchik,M.J。(1996)。 疾病相关的类风湿因子自身抗体在正常小鼠中不耐受:对自身抗体来源的影响in autoimmune disease。 J Exp Med 184(4):1269-1278。
              3. Prak,E.L.和Weigert,M。(1995)。 轻链替换:抗体基因重排的新模型。 J Exp Med 182(2):541-548。
              4. Shlomchik,M.J.,Zharhary,D.,Saunders,T.,Camper,S.A。和Weigert,M.G。(1993)。 自身抗体调节的类风湿因子转基因小鼠模型。 Int Immunol 5(10):1329-1341。
              5. Sonoda,E.,Pewzner-Jung,Y.,Schwers,S.,Taki,S.,Jung,S.,Eilat,D.and Rajewsky,K。 等位基因包含条件下的B细胞发育。 免疫力 6(3):225-233。
              6. Sweet,R.A.,Cullen,J.L。和Shlomchik,M.J。(2013)。 类风湿因子B细胞记忆导致快速,转换的抗体形成细胞反应。 em Immunol 190(5):1974-1981。
              7. Tomayko,M.M.,Steinel,N.C.,Anderson,S.M.and Shlomchik,M.J.(2010)。 切缘:鼠记忆B细胞亚群的成熟度层次。 Immunol 185(12):7146-7150。
              8. Zuccarino-Catania,G.V.,Sadanand,S.,Weisel,F.J.,Tomayko,M.M.,Meng,H.,Kleinstein,S.H.,Good-Jacobson,K.L.and Shlomchik,M.J.(2014)。 CD80和PD-L2定义独立于抗体同种型的功能上不同的记忆B细胞亚群。 a> Nat Immunol 15(7):631-637。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zuccarino-Catania, G. and Shlomchik, M. (2015). Adoptive Transfer of Memory B Cells. Bio-protocol 5(16): e1563. DOI: 10.21769/BioProtoc.1563.
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