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In vitro Assessment of Immunological Synapse Formation by Flow Cytometry
流式细胞法体外评估免疫突触的形成   

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Abstract

In adaptive immune system, formation of immunological synapse between T cells and antigen presenting cells (dendritic cells, B cells, and macrophages) or target cells (tumor cells and viral-infected cells) is critical for the execution of T cell immune responses via cytokine secretion or direct killing activity. Here, we describe the practical methods that directly measure the number of conjugates as a result of immunological synapse formation between T cells and superantigen-loaded B cells or between cytotoxic T cells and antigen-loaded target cells by dual-color flow cytometry.

Keywords: T cells(T细胞), Antigen-presenting cells(抗原提呈细胞), Immunological synapse(免疫突触), Adhesion(粘附), Conjugation(共轭)

Materials and Reagents

  1. 15 ml and 50 ml conical tube
  2. 12 well cell culture plate
  3. FalconTM Cell strainer (40 μm Nylon) (Corning, catalog number: 352340 )
  4. 1 ml syringe
  5. FalconTM 5 ml Polystyrene Round-Bottom tube (Corning, catalog number: 352052 )
  6. MACS MS column (Miltenyi Biotec, catalog number: 130-042-201 ) and MACS separator (Miltenyi Biotec)
  7. Jurkat T cell (ATCC, catalog number: TIB-152 )
  8. Raji B cell (ATCC, catalog number: CCL-86 )
  9. EL4 cell (ATCC, catalog number: TIB-39 )
  10. C57BL/6 mice (8-10 weeks)
  11. OTI mice (8-10 weeks) (THE JACKSON LABORATORY, catalog number: 021773 )
    Note: These mice have the transgenic T cell receptor designed to recognize ovalbumin residues 257-264 in the context of H2Kb.
  12. Staphylococcus Enterotoxin E (SEE) (Toxin Technology, catalog number: ET404 )
  13. Staphylococcus Enterotoxin B (SEB) (Toxin Technology, catalog number: BT202 )
  14. OVA 257–264 peptide (InvivoGen, catalog number: vac-sin )
  15. CellTracker Green CMFDA (Life Technologies, Molecular Probes®, catalog number: C7025 )
    Note: Currently, it is “Thermo Fisher Scientific, Molecular ProbesTM, catalog number: C7025”.
  16. CellTracker Orange CMRA (Life Technologies, Molecular Probes®, catalog number: C34551 )
    Note: Currently, it is “Thermo Fisher Scientific, Molecular ProbesTM, catalog number: C34551”.
  17. RBC lysis buffer (BioLegend, catalog number: 42030 )
  18. Mouse CD3+ T cell enrichment column kit (R&D Systems, catalog number: MTCC535 )
  19. Mouse B cell enrichment kit (STEMCELL Technologies, catalog number: 19754A )
  20. CD8a (Ly-2) MicroBeads (Miltenyi Biotec, catalog number: 130-049-401 )
  21. Recombinant human IL-2 (rhIL-2) (NIH AIDS Reagent Program, catalog number: 11697 )
  22. RPMI1640 (Thermo Fisher Scientific, GibcoTM, catalog number: 31800 )
  23. 10% heat-inactivated fetal bovine serum (FBS) (Siyaku, Wako Pure Chemical Industries, AusGeneX, catalog number: FBSUS500 )
  24. Penicillin/streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140 )
  25. Non-essential amino acids (Thermo Fisher Scientific, GibcoTM, catalog number: 11140 )
  26. Sodium pyruvate (Thermo Fisher Scientific, GibcoTM, catalog number: 11360 )
  27. 2-Mercaptoethanol (Sigma-Aldrich, catalog number: M-7522 )
  28. Complete RPMI1640 medium (see Recipes)
  29. Mouse T cell medium (see Recipes)

Equipment

  1. Water Jacketed CO2 incubator (37 °C, 5% CO2) (Thermo Fisher Scientific)
  2. FACSCanto instrument (BD Bioscience)

Procedure

  1. Conjugation assay using cell lines
    1. Incubate Jurkat T cells (2 x 105/sample) with 1 μM of CellTracker Green CMFDA and Raji B cells (2 x 105/sample) with 3 μM CellTracker Orange CMRA in 0.5-1 ml of complete RPMI1640 medium in 15 ml conical tubes for 30 min at 37 °C in a CO2 incubator.
      Note: Perform all steps in the dark to prevent bleaching of fluorescence.
    2. For washing, centrifuge the cells at 1,300 rpm for 3 min, discard the supernatant, and resuspend cell pellet using complete RPMI1640 medium. Repeat twice. After washing, resuspend the cells in 200 μl/sample of complete RPMI1640 medium.
    3. Treat CMRA-labelled Raji B cells in 200 μl/sample of complete RPMI1640 medium with 5 μg/ml SEE for 30 min at 37 °C.
      Notes:
      1. SEE and SEB (B.5) act as the superantigens. Prepare SEE and SEB stock solution of 1 mg/ml in sterile distilled water (SDW) and store in -80 °C.
      2. Control samples of conjugation assay are samples untreated with SEE, SEB (B), and OVA peptide (C).
      3. SEE and SEB act as a superantigen that cause non-specific activation of T cells resulting in polyclonal T cell activation and massive cytokine release. Although SEE and SEB can bind both TCR and MHC to induce T cell activation, binding sites are different. SEE is used for formation of conjugation between Jurkat T cell-Raji B cell because Jurkat T cells have the human TCR Vβ recognized by SEE, not SEB. SEE can also apply to form mouse T cell-B cell conjugates, but SEB is more widely used in mouse T cell activation.
    4. Mix and incubate equal cell number of CMFDA-T cells (2 x 105/200 μl/sample) and CMRA-B cells (2 x 105/200 μl/sample) in complete RPMI1640 medium in the polystyrene round-bottom tube for 30 min at 37 °C in a CO2 incubator.
    5. Analyze the T cell-B cell conjugates using dual-color flow cytometry.
      1. Prepare the samples as the table below.

        Unstained
        Single-Color
        Dual-Color
        T cells
        B cells/EL4 cells
        CMFDA (green) labeled T cells
        CMRA (red) labeled B/EL4 cells
        CMFDA (green) labeled T cells co-incubated CMRA (orange) labeled B/EL4 cells with or without treatment of SEE/SEB/OVA
        Set aside 2 x 105 of these cells before starting step A1 for step A5b
        Set aside 2 x 105 of these cells before starting step A1 for step A5b
        Set aside 2 x 105 of these cells before step A3 for step A5c
        Set aside 2 x 105 of these cells before step A3 for step A5c
        These samples will be used for step A5d

      2. Analyze unstained control to set voltage and gates (FSC, SSC, fluorescence signals).
      3. Analyze single-color stained controls for compensation.
      4. After compensation, analyze dual-color stained samples.
        Notes:
        1. Read the fluorescence of 10,000-20,000 cells in all controls and samples for analysis.
        2. The percentage of green-orange events indicates the proportion of T cell-B cell conjugates (Figure 1).

  2. Conjugation assay using mouse T cells and B cells
    1. Isolate splenocytes from the spleen from a C57BL/6 mouse.
      1. Cut out the spleen.
      2. Mash the spleen through the cell strainer into 50 ml conical tube using the plunger end of the 1 ml syringe.
      3. Wash the cell strainer with 5-6 ml mouse T cell medium and discard the strainer.
      4. Transfer the suspended cells into a 15 ml conical tube, and spin down cells at 1,800 rpm for 3 min.
      5. Discard supernatant and resuspend cell pellet in 1 ml RBC (Red blood cell) lysis buffer to remove RBCs. After 1 min, add 5 ml mouse T cell media and discard any dead cell mass.
      6. Spin down at 1,800 rpm for 3 min, and discard supernatant. Resuspend pellet in T cell enrichment buffer provided by the manufacturer or B cell enrichment buffer (2% FBS in PBS).
    2. Isolate CD3+ T cells from splenocytes using Mouse CD3+ T cell enrichment column kit according to the manufacturer’s protocol. Isolate B cells from mouse splenocytes using Mouse B cell enrichment kit according to the manufacturer’s protocol. After isolation, resuspend cells in mouse T cell medium.
      Note: The yield of T cell and B cell enrichment are above 95%.
    3. Incubate CD3+ T cells with 1 μM of CellTracker Green CMFDA (2 x 105/sample) and B cells with 3 μM CellTracker Orange CMRA (2 x 105/sample) in 0.5-1 ml of mouse T cell medium at 37 °C in 15 ml conical tubes as described in step A1.
    4. For washing, centrifuge the cells at 1,800 rpm for 3 min, discard the supernatant, and resuspend cell pellet using mouse T cell medium. Repeat twice. After washing, resuspend the cells in 200 μl/sample of mouse T cell medium.
    5. Treat B cells with 5 μg/ml SEB in 200 μl/sample of mouse T cell medium for 30 min at 37 °C.
    6. Mix and incubate equal cell number in 200 μl/sample of mouse T cell medium of CMFDA-CD3+ T cells and CMRA-B cells in the polystyrene round-bottom tube for 30 min at 37 °C in a CO2 incubator.
    7. Analyze the T cell-B cell conjugates using dual-color flow cytometry as described in step A5.

  3. Conjugation assay using antigen-specific cytotoxic T cells and target cells
    1. Isolate splenocytes from an OTI mouse as described in step B1, and resuspend splenocytes in mouse T cell medium.
      Note: CD8+ T cells purified from an OTI-transgenic TCR mice express Ova-specific TCR that can recognize OVA 257-264 peptide as an antigen.
    2. For differentiation to OVA-specific cytotoxic T cells, stimulate splenocytes (2 x 106 cells/well) with OVA 257-264 peptide in mouse T cell medium with 10 U/ml rhIL-2 in 12 well cell culture plates for 2-3 days. Add rhIL-2 to media daily.
      Note: Thaw frozen stock of rhIL-2 (kept at -80 °C) and dilute in mouse T cell medium. Diluted rhIL-2 can be stored at 4 °C for one month.
    3. Isolate OVA-specific cytotoxic T cells by MACS separation using CD8a (Ly-2) MicroBeads according to the manufacturer’s protocol.
    4. Incubate OVA-specific cytotoxic T cells with 1 μM of CellTracker Green CMFDA (2 x 105-1 x 106/sample) and EL4 cells (as target cells; 2 x 105/sample) with 3 μM CellTracker Orange CMRA at 37 °C in mouse T cell medium, wash, and resuspend in mouse T cell medium as described in steps B3-4.
      Note: The relevant ratio of cytotoxic T cells and EL4 cells is 1:1-5:1.
    5. Add 10 μg/ml OVA 257–264 peptide to EL4 cells in 200 μl/sample of mouse T cell medium for 30 min at 37 °C.
      Note: OVA peptide loaded on EL4 cells acts as the antigen recognized by OVA-specific cytotoxic T cells.
    6. Mix and incubate equal cell number in 200 μl/sample of mouse T cell medium of CMFDA-cytotoxic T cells and CMRA-EL4 cells in the polystyrene round-bottom tube for 30 min-1 h at 37 °C in a CO2 incubator.
    7. Analyze the cytotoxic T cell-target cell conjugates using dual-color flow cytometry as described in step A5.

Representative data


Figure 1. SEE-induced Jurkat T cell-Raji B cell conjugates formation. Conjugates formation using Jurkat T cells and Raji B cells with or without SEE were performed as described in Procedure section A and then analyzed by flow cytometry. Dot plot showing FSC and SSC is gated to exclude cell debris and select cells for dual-color analysis (Left). Cells inside the gate were analyzed by CMFDA (green; T cell) and CMRA (red; B cell) fluorescence signals (middle dot plots). Quadrants are established using negative control and single-color controls. CMFDA+ CMRA- cells (lower right quadrant) indicate T cell only, CMFDA- CMRA+ cells (upper left quadrant) indicate B cell only, and CMFDA+ CMRA+ cells (upper right quadrant) indicate T cell-B cell conjugates. After loading of SEE by Raji B cells (+SEE), T cell-B cell conjugates were increased in compared with SEE-unloaded B cells (-SEE). Numbers in dot plots indicate the percentage of each quadrant. Confocal fluorescence image and differential interference contrast (DIC) image show T cell only (green box), B cell only (red box), and T cell-B cell conjugate (yellow box) after conjugation assay with SEE (Right).

Recipes

  1. Complete RPMI1640 medium
    RPMI1640
    10% heat-inactivated fetal bovine serum
    100 U/ml Penicillin/streptomycin
  2. Mouse T cell medium
    RPMI1640
    10% heat-inactivated fetal bovine serum
    100 U/ml Penicillin/streptomycin
    1% non-essential amino acids
    1 mM Sodium pyruvate
    50 μM 2-Mercaptoethanol

Acknowledgments

This work was supported by the Basic Science Program (2015R1A2A1A15052658), and the Creative Research Initiative Program (2015R1A3A2066253) through the National Research Foundation (NRF) grants funded by the Ministry of Science, ICT & Future Planning (MSIP), Korea, and Bioimaging Research Center at GIST.

References

  1. Na, B. R., Kim, H. R., Piragyte, I., Oh, H. M., Kwon, M. S., Akber, U., Lee, H. S., Park, D. S., Song, W. K., Park, Z. Y., Im, S. H., Rho, M. C., Hyun, Y. M., Kim, M. and Jun, C. D. (2015). TAGLN2 regulates T cell activation by stabilizing the actin cytoskeleton at the immunological synapse. J Cell Biol 209(1): 143-162.

简介

在适应性免疫系统中,T细胞和抗原呈递细胞(树突细胞,B细胞和巨噬细胞)或靶细胞(肿瘤细胞和病毒感染细胞)之间的免疫突触的形成对通过细胞因子执行T细胞免疫应答至关重要 分泌或直接杀死活动。 在这里,我们描述了通过双色流式细胞术直接测量T细胞和加载超抗原的B细胞之间或细胞毒性T细胞和抗原负载的靶细胞之间的免疫突触形成的结合物数量的实际方法。

关键字:T细胞, 抗原提呈细胞, 免疫突触, 粘附, 共轭

材料和试剂

  1. 15 ml和50ml锥形管
  2. 12孔细胞培养板
  3. Falcon TM细胞过滤器(40μm尼龙)(Corning,目录号:352340)
  4. 1 ml注射器
  5. Falcon TM 5ml聚苯乙烯圆底管(Corning,目录号:352052)
  6. MACS MS柱(Miltenyi Biotec,目录号:130-042-201)和MACS分离器(Miltenyi Biotec)
  7. Jurkat T细胞(ATCC,目录号:TIB-152)
  8. Raji B细胞(ATCC,目录号:CCL-86)
  9. EL4细胞(ATCC,目录号:TIB-39)
  10. C57BL/6小鼠(8-10周)
  11. OTI小鼠(8-10周)(THE JACKSON LABORATORY,目录号:021773)
    注意:这些小鼠具有转基因T细胞受体,其被设计为在H2Kb的背景中识别257-264的卵白蛋白残基。
  12. 葡萄球菌肠毒素 E(SEE)(Toxin Technology,目录号:ET404)
  13. 葡萄球菌肠毒素B(SEB)(Toxin Technology,目录号:BT202)
  14. OVA 257-264肽(InvivoGen,目录号:vac-sin)
  15. CellTracker Green CMFDA(Life Technologies,Molecular Probes ,目录号:C7025)
    注意:目前,"Thermo Fisher Scientific,Molecular Probes TM ,目录号:C7025" br />
  16. CellTracker Orange CMRA(Life Technologies,Molecular Probes ,目录号:C34551)
    注意:目前,"Thermo Fisher Scientific,Molecular Probes TM ,目录号:C34551" br />
  17. RBC裂解缓冲液(BioLegend,目录号:42030)
  18. 小鼠CD3 + T细胞富集柱试剂盒(R& D Systems,目录号:MTCC535)
  19. 小鼠B细胞富集试剂盒(STEMCELL Technologies,目录号:19754A)
  20. CD8a(Ly-2)MicroBeads(Miltenyi Biotec,目录号:130-049-401)
  21. 重组人IL-2(rhIL-2)(NIH AIDS Reagent Program,目录号:11697)
  22. RPMI1640(Thermo Fisher Scientific,Gibco TM ,目录号:31800)
  23. 10%热灭活的胎牛血清(FBS)(Siyaku,Wako Pure Chemical Industries,AusGeneX,目录号:FBSUS500)
  24. 青霉素/链霉素(Thermo Fisher Scientific,Gibco TM ,目录号:15140)
  25. 非必需氨基酸(Thermo Fisher Scientific,Gibco TM ,目录号:11140)
  26. 丙酮酸钠(Thermo Fisher Scientific,Gibco< sup> TM,目录号:11360)
  27. 2-巯基乙醇(Sigma-Aldrich,目录号:M-7522)
  28. 完成RPMI1640介质(参见配方)
  29. 小鼠T细胞培养基(见配方)

设备

  1. 水夹套CO 2培养箱(37℃,5%CO 2)(Thermo Fisher Scientific)
  2. FACSCanto仪器(BD Bioscience)

程序

  1. 使用细胞系的缀合测定
    1. 将Jurkat T细胞(2×10 5个/样品)与1μM的CellTracker Green ?CMFDA和Raji B细胞(2×10 5个/样品)与3μMCellTracker Orange CMRA在0.5-1ml完全RPMI1640培养基的15ml锥形管中 37℃下在CO 2培养箱中孵育30分钟 注意:在黑暗中执行所有步骤,以防止荧光漂白。
    2. 对于洗涤,以1,300rpm离心细胞3分钟,弃去 上清液,并使用完全RPMI1640重悬细胞沉淀 中。重复两次。洗涤后,在200中重悬细胞 μl/完全RPMI1640培养基的样品
    3. 将CMRA标记的Raji B细胞在37℃下在200μl/含有5μg/ml SEE的完全RPMI1640培养基的样品中处理30分钟。 注意:
      1. SEE和SEB(B.5)充当超级抗原。准备SEE和SEB 储存在无菌蒸馏水(SDW)中的1mg/ml储存溶液, -80°C。
      2. 缀合测定的对照样品是未用SEE,SEB(B)和OVA肽(C)处理的样品。
      3. SEE和SEB充当引起非特异性激活的超抗原 ?的T细胞导致多克隆T细胞活化和大量 细胞因子释放。虽然SEE和SEB可以结合TCR和MHC两者 诱导T细胞活化,结合位点不同。 SEE用于 Jurkat T细胞 - Raji B细胞之间形成共轭 Jurkat T细胞具有由SEE识别的人TCRVβ,而不是SEB。 SEE可以 ?也适用于形成小鼠T细胞-B细胞结合物,但SEB更多 广泛应用于小鼠T细胞活化。
    4. 混合和孵育平等 CMFDA-T细胞(2×10 5/200 /200μl/样品)的细胞数和CMRA-B细胞(2 ?x 10 /200μl/样品)在聚苯乙烯的完全RPMI1640培养基中 圆底管在37℃下在CO 2培养箱中孵育30分钟。
    5. 使用双色流式细胞术分析T细胞-B细胞缀合物
      1. 按下表准备样品。

        未清除
        单色
        双色
        T细胞
        B细胞/EL4细胞 CMFDA(绿色)标记的T细胞
        CMRA(红色) 标记的B/EL4细胞
        CMFDA(绿色)标记的T细胞共孵育CMRA (橙色)标记的B/EL4细胞,用或不用SEE/SEB/OVA处理
        在开始步骤A5b的步骤A1之前,将2×10 5个 在开始步骤A5b的步骤A1之前,将2×10 5个 在步骤A5之前,将这些单元格中的2 x 10 5 搁置一边
        搁置 2×10 5 个步骤 这些样品将 用于步骤A5d

      2. 分析未染色的控制以设置电压和门(FSC,SSC,荧光信号)
      3. 分析单色染色控制以获得补偿。
      4. 补偿后,分析双色染色样品 注意:
        1. 读取所有对照和样品中10,000-20,000个细胞的荧光,用于分析。
        2. 绿 - 橙事件的百分比表示T细胞-B细胞缀合物的比例(图1)。

  2. 使用小鼠T细胞和B细胞的缀合测定
    1. 分离来自C57BL/6小鼠脾脏的脾细胞
      1. 切除脾脏。
      2. 使用1ml注射器的柱塞端将脾脏通过细胞过滤器捣碎到50ml锥形管中
      3. 用5-6ml小鼠T细胞培养基洗细胞滤网,弃去过滤器
      4. 将悬浮的细胞转移到15ml锥形管中,并以1,800rpm离心细胞3分钟
      5. 弃去上清液并将细胞沉淀重悬在1ml RBC中(红血 ?细胞)裂解缓冲液以除去RBC。 1分钟后,加入5毫升小鼠T细胞 介质并丢弃任何死细胞团块
      6. 在1,800 rpm下旋转3 ?min,弃去上清液。在T细胞富集中重悬沉淀 缓冲液或B细胞富集缓冲液(2%FBS 在PBS中)。
    2. 分离来自脾细胞的CD3 + T细胞,使用小鼠CD3 sup T ?细胞富集柱试剂盒根据制造商的方案。 使用小鼠B细胞富集试剂盒从小鼠脾细胞分离B细胞 ?根据制造商的协议。分离后,重悬 小鼠T细胞培养基中的细胞 注意:T细胞和B细胞富集的产率高于95%。
    3. 用1μMCellTracker Green CMFDA(2×10 6细胞)孵育CD3 + T细胞 10 /样品)和B细胞用3μMCellTracker Orange CMRA(2× 10 /样品)在0.5-1ml的小鼠T细胞培养基中在37℃下在15ml圆锥形中 ?管,如步骤A1中所述
    4. 对于洗涤,离心细胞 ?在1,800rpm离心3分钟,弃去上清液,并重悬细胞 使用小鼠T细胞培养基。重复两次。洗涤后,重悬 ?细胞在200μl/样品的小鼠T细胞培养基中
    5. 在37℃下用200μl/小鼠T细胞培养基样品中的5μg/ml SEB处理B细胞30分钟。
    6. 混合和孵育等量细胞在200微升/样本的小鼠T细胞 在聚苯乙烯中的CMFDA-CD3 + T细胞和CMRA-B细胞的培养基 圆底管在37℃下在CO 2培养箱中孵育30分钟。
    7. 使用如步骤A5中所述的双色流式细胞术分析T细胞-B细胞缀合物。

  3. 使用抗原特异性细胞毒性T细胞和靶细胞的缀合测定
    1. 如步骤B1所述分离来自OTI小鼠的脾细胞,并在小鼠T细胞培养基中重悬脾细胞。
      从OTI转基因TCR小鼠中纯化的CD8 + Ova特异性TCR,其可识别OVA 257-264肽作为抗原。
    2. 为分化为OVA特异性细胞毒性T细胞,刺激 脾细胞(2×10 6个细胞/孔)与小鼠T中的OVA 257-264肽 细胞培养基与10 U/ml rhIL-2在12孔细胞培养板中2-3 天。每天向媒体添加rhIL-2。
      注意:解冻rhIL-2的冷冻储备液 (保持在-80℃)并在小鼠T细胞培养基中稀释。稀释rhIL-2罐 在4℃保存一个月。
    3. 分离OVA特异性细胞毒性T ?细胞通过MACS分离使用CD8a(Ly-2)MicroBeads根据 制造商协议
    4. 孵育OVA特异性细胞毒性T细胞 与1μM的CellTracker Green CMFDA(2×10 5 -1×10 6 /样品)和EL4 细胞(作为靶细胞; 2×10 5个/样品)与3μMCellTracker Orange混合 CMRA在37℃下在小鼠T细胞培养基中洗涤,并重悬于小鼠T中 细胞培养基,如步骤B3-4中所述 注意:细胞毒性T细胞和EL4细胞的相关比率为1:1-5:1。
    5. 在37℃下,在200μl/小鼠T细胞培养基样品中向EL4细胞中加入10μg/ml OVA 257-264肽30分钟。 注意:装载在EL4细胞上的OVA肽作为由OVA特异性细胞毒性T细胞识别的抗原。
    6. 混合和孵育等量细胞在200微升/样本的小鼠T细胞 CMFDA-细胞毒性T细胞和CMRA-EL4细胞的培养基 圆底管在37℃下在CO 2培养箱中孵育30分钟-1小时。
    7. 使用如步骤A5中所述的双色流式细胞术分析细胞毒性T细胞 - 靶细胞缀合物。

代表数据


图1.SEE诱导的Jurkat T细胞 - Raji B细胞缀合物形成。 如过程部分A所述,使用Jurkat T细胞和具有或不具有SEE的Raji B细胞进行缀合物形成,然后通过流式细胞术进行分析。显示FSC和SSC的点图被选通以排除细胞碎片并选择用于双色分析的细胞(左图)。通过CMFDA(绿色; T细胞)和CMRA(红色; B细胞)荧光信号(中间点图)分析门内部的细胞。使用负控制和单色控制建立象限。 CMFDA + CMRA - 细胞(右下象限)表示仅T细胞,CMFDA - 左象限)表示仅B细胞,CMFDA + CMRA + 细胞(右上象限)表示T细胞-B细胞缀合物。在加载Raji B细胞(+ SEE)的SEE后,与SEE-未加载的B细胞(-SEE)相比,T细胞-B细胞缀合物增加。点状图中的数字表示每个象限的百分比。共聚焦荧光图像和微分干涉对比(DIC)图像显示与SEE(右)缀合测定后的仅T细胞(绿色框),仅B细胞(红色框)和T细胞-B细胞结合物(黄色框)。

食谱

  1. 完成RPMI1640介质
    RPMI1640
    10%热灭活的胎牛血清 100U/ml青霉素/链霉素
  2. 小鼠T细胞培养基
    RPMI1640
    10%热灭活的胎牛血清 100U/ml青霉素/链霉素 1%非必需氨基酸
    1mM丙酮酸钠 50μM2-巯基乙醇

致谢

这项工作由基础科学计划(2015R1A2A1A15052658)和创新研究计划(2015R1A3A2066253)通过国家研究基金会(NRF)资助的科学,信息和通信技术部未来规划(MSIP),韩国,以及GIST的生物成像研究中心。

参考文献

  1. Na,BR,Kim,HR,Piragyte,I.,Oh,HM,Kwon,MS,Akber,U.,Lee,HS,Park,DS,Song,WK,Park,ZY, Hyun,YM,Kim,M。和Jun,CD(2015)。 TAGLN2通过稳定免疫突触的肌动蛋白细胞骨架来调节T细胞活化。 J Cell Biol 209(1):143-162。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Na, B. and Jun, C. (2016). In vitro Assessment of Immunological Synapse Formation by Flow Cytometry. Bio-protocol 6(6): e1758. DOI: 10.21769/BioProtoc.1758.
  2. Na, B. R., Kim, H. R., Piragyte, I., Oh, H. M., Kwon, M. S., Akber, U., Lee, H. S., Park, D. S., Song, W. K., Park, Z. Y., Im, S. H., Rho, M. C., Hyun, Y. M., Kim, M. and Jun, C. D. (2015). TAGLN2 regulates T cell activation by stabilizing the actin cytoskeleton at the immunological synapse. J Cell Biol 209(1): 143-162.
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