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In vitro T Cell–DC and T Cell–T Cell Clustering Assays

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Nature Immunology
Apr 2013



To get activated, T cells need to find their cognate antigen at the surface of an antigen-presenting cell (APC). Recognition of cognate antigen in the context of the MHC (Major histocompatibility complex) by the TCR (T-Cell Receptor) results in long lasting interactions between T cells and APCs. Subsequently, T cells form homotypic interactions with each other, which is seen as a hallmark of T cell activation. This protocol describes a method to analyze T-APC and T-T conjugation.

Materials and Reagents

  1. RPMI-1640 medium (Life Technologies, Gibco®, catalog number: 11875-093 )
  2. FBS (Hyclone, catalog number: SH30396-03 )
  3. Penicillin-Streptomycin-Glutamine Solution (Life Technologies, Gibco®, catalog number: 10378-016 )
  4. 2-mercaptoethanol (Life Technologies, Invitrogen™, catalog number: 21985-023 )
  5. Phorbol 12-myristate-13-acetate (PMA) (Sigma-Aldrich, catalog number: 79346 )
  6. Ionomycin (Sigma-Aldrich, catalog number: 19657 )
  7. 5-(and-6)-Carboxyfluorescein Diacetate, Succinimidyl Ester (5(6)-CFDA, SE) (CFSE) (Life Technologies, Molecular Probes®, catalog number: C1157 )
  8. 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one (DDAO) (Life Technologies, Invitrogen™, catalog number: C34553 )
  9. 5-(and-6)-(((4-Chloromethyl)Benzoyl)Amino) Tetramethylrhodamine (CMTMR) (Life Technologies, Invitrogen™, catalog number: C2927 )
  10. 16% Paraformaldehyde Solution (PFA) (16% Formaldehyde, EM Grade) (Electron Microscopy Sciences, catalog number: 15710 )
  11. Vectashield Hardset with Dapi (VWR International, catalog number: 101098-050 )
  12. Poly-L-Lysine Solution 0.1% (w/v) in H2O (Sigma-Aldrich, catalog number: P8920 )
  13. SL8 (Ovalbumin (257-264)) (AnaSpec, catalog number: 60193 )
  14. PBS (Life Technologies, catalog number: 14190-44 )
  15. Media (see Recipes)
  16. 2% PFA (see Recipes)
  17. Poly-L-Lysine-coated chamber (see Recipes)


  1. Centrifuge
  2. Flow Cytometer
  3. Epifluorescent microscope
  4. 37 °C, 5% CO2 Cell culture incubator
  5. 24 well plate (Corning, Costar®, catalog number: 3524 )
  6. 40 μm Cell strainer (BD Biosciences, Falcon®, catalog number: 352340 )
  7. FACS tube, polystyrene (BD Biosciences, Falcon®, catalog number: 352054 )
  8. 8-well chamber slide (Thermo Fisher Scientific, Lab-TekTM, catalog number: 177445 )


  1. T-APC conjugation assay
    1. This protocol uses purified OT-I CD8+ T-cells from naive mice, which are transgenic for a TCR recognizing the chicken egg OVA–derived SIINFEKL peptide (SL8) in the context of the MHC class I molecule H2-kb. The same protocol can be used with T cells specific for other antigens. BMDCs (Bone-Marrow derived Dendritic Cells) are used as APCs, but DCs from other origin, or many cell lines, can be used as APCs.
    2. Once cells are labeled, they should stay protected from light as much as possible.

    1. Cell labelling: resuspend BMDCs and purified naive OT-I cells in PBS at a concentration of 10 x 106 cells/ml (usually 20 x 106 cells in 2 ml). Add DDAO at a final concentration of 4 μM on BMDCs, and CFSE at a final concentration of 1 μM on OT-I cells. Incubate for 30 min at 37 °C 5% CO2.
      Note: CFSE and DDAO can be substituted for other dyes. The advantage using those 2 dyes is that little to no compensation on the flow cytometer is needed.
    2. Wash cells three times with 5 volumes of media (10 ml if you start with 20 x 106 cells) by centrifuging at 300 x g for 5 min at room temperature.
    3. Pulse APCs: resuspend BMDCs in media at a concentration of 10 x 106 cells/ml. Add SL8 at a final concentration ranging from 0-1,000 ng/ml (usually 5 different concentrations are used) and incubate for 60 min at 37 °C 5% CO2.
    4. While APCs are antigen pulsing, resuspend OT-I cells at 4 x 106 cells/ml in media (usually 20 x 106 cells in 5 ml). Add 50 μl of OT-I cells per FACS tube. Leave cells at 37 °C 5% CO2 until APCs are ready.
    5. Wash BMDCs three times with 5 volumes of media by centrifuging at 300 x g for 5 min at room temperature.
    6. Resuspend BMDCs at 4 x 106 cells/ml in media.
    7. Add 50 μl of BMDCs to each FACS tube containing OT-I cells. Immediately spin at 228 x g (1,000 rpm) for 1 min at 20 °C. Immediately place at 37 °C.
      Note: In this protocol, the ratio T cell-APC is 1, but you can make it vary. Time of incubation is usually between 0-60 min (0-10-20-30-60 min).
    8. Fix the cells: Once the desired amount of time is completed, add 100 μl per sample of warm (20 °C) 2% PFA. Mix very gently, either by pipetting up and down or quickly vortexing on low. Incubate at room temperature (around 20 °C) for 10 min.
    9. Run on flow cytometer. Assess the percentage of coupling based on double CFSE/DDAO positives (Figure 1).

      Figure 1. T-APC coupling assay-Example.
      CFSE-labeled OT-I cells are incubated for 30 min at 37 °C with DDAO-labeled BMDCs that were pulsed with 0 (left plot) or 100 ng/ml (right plot) SL8 peptide. Cells debris were first excluded using a SSC/FSC gate (upper panel). Coupled cells are DDAO+ and CFSE+.

  2. T-T clustering assay
    1. In this protocol, T cells are activated with PMA and Ionomycin, which by-pass the need for an APC and every T cell could be activated this way. However, the same protocol can be used with T cells activated with an APC, anti-CD3 and anti-CD28, etc...
    2. This protocol compares, as an example, the capacity of OT-I cells deficient for the integrin ICAM-1 to form homotypic interactions compared to control OT-I cells. But the same protocol can be used to investigate the kinetic of T cell clustering, blocking antibodies, etc…

    1. Cell labelling: resuspend purified OT-I cells in PBS at a concentration of 10 x 106 cells/ml (usually 20 x 106 cells in 2 ml). Add CFSE at a final concentration of 1 μM on Control cells, and CMTMR at a final concentration of 4 μM on ICAM-1-/- cells. Incubate for 30 min at 37 °C 5% CO2.
    2. Wash cells three times with 5 volumes of media by centrifuging at 300 x g for 5 min at room temperature.
    3. T cell activation: Resuspend OT-I cells in media at a concentration of 2 x 106 cells/ml. WT and ICAM-1-/- cells are mixed at a ratio 1 and then activate cells by adding 5 ng/ml PMA and 50 ng/ml ionomycin directly in the media. Plate 2 ml cell suspension per well of a 24 well plate.
    4. 16 to 24 h after activation, percentage of cells in clustered is analyzed by flow cytometry (step B5) or microscopy (step B6).
    5. Analysis by flow cytometry: Cells are fixed by adding the same volume of 2% PFA for 10 min at 20 °C. Fixed cells are run through a 40 μm strainer into a FACS tube. Cells that went through represent the “non-clustered” fraction. Cells retained on the strainer are washed with 1 ml of PBS 2 mM EDTA (to disrupt cell clusters) and represent the “clustered” fraction. Both fractions are run on flow cytometer (Figure 2).

      Figure 2. T-T clustering assay–Analysis by flow cytometry. CFSE-labeled WT OT-I cells and CMTMR-labeled ICAM-1-/- OT-I cells were ad-mixed and stimulated with PMA and Ionomycin. 24 h after activation, clustered cells were separated from non-clustered cells through a 40 μm strainer and both fractions were analyzed by flow cytometry. Cell debris was first excluded using a SSC/FSC gate (upper panel). Clustered cells – left panel; non-clustered cells – right panel.
      Note that ICAM-1 deficient cells are mainly found in the non-clustered cell fraction compared to WT cells.
      Note: Cut the tip of your pipet tip to ensure that cell clusters are not disrupted by pipetting when applying the cell suspension on the strainer.
      In vitro activated T cells usually form clusters that are bigger than 40 μm diameter and therefore should be retained on the strainer. However, cells that are going through the strainer can potentially be part of smaller clusters. It is therefore useful to compare the flow cytometry data with microscopy quantification.

    6. Analysis by microscopy: Cells (usually 300 μl of cell suspension) are transferred onto a poly-L-Lysine-coated chamber and incubated for 10 min at 37 °C 5% CO2. Media is then carefully removed and replaced by 200 μl of 2% PFA. After 10 min, PFA is carefully removed, and replaced by 200 μl of PBS. Remove the PBS and let dry for 2 min. Remove the chamber gasket and mount with a drop of Vectashield on a coverslip. Analyze with epifluorescent microscope.
      Note: Cut the tip of your pipet tip to ensure that cell clusters are not disrupted by pipetting when applying the cell suspension on the chamber.


  1. Media (555 ml)
    500 ml RPMI Medium 1640
    50 ml heat inactivated (30 min at 56 °C) FCS
    500 μl of 50 mM β-mercaptoethanol
    5 ml of Penicillin-Streptomycin-Glutamine Solution
    Keep at 4 °C
  2. 2% PFA
    Dilute 1 ml of 16% PFA solution in 7 ml PBS
    Keep in dark at 4 °C
  3. Poly-L-Lysine coated chambers
    Dilute poly-L-Lysine stock solution 1/100 in dH2O
    Put 500 μl on each chamber
    Incubate 5-10 min at RT
    Wash thoroughly with dH2O and let the chambers dry at least 2 h
    Chambers can be stocked for several weeks at RT


This protocol was adapted from the previously published paper Gerard et al. (2013). This protocol was adapted in the laboratory of Matthew F. Krummel, and was supported by grants from the Juvenile Diabetes Foundation (MFK), and NIH R01AI52116 (MFK).


  1. Gérard, A., Khan, O., Beemiller, P., Oswald, E., Hu, J., Matloubian, M. and Krummel, M. F. (2013). Secondary T cell-T cell synaptic interactions drive the differentiation of protective CD8+ T cells. Nat Immunol 14(4): 356-363.


为了激活,T细胞需要在抗原呈递细胞(APC)的表面找到它们的同源抗原。 通过TCR(T细胞受体)在MHC(主要组织相容性复合物)的上下文中的同源抗原的识别导致T细胞和APC之间的持久的相互作用。 随后,T细胞彼此形成同型相互作用,这被看作是T细胞活化的标志。 该协议描述了一种分析T-APC和T-T共轭的方法。


  1. RPMI-1640培养基(Life Technologies,Gibco ,目录号:11875-093)
  2. FBS(Hyclone,目录号:SH30396-03)
  3. 青霉素 - 链霉素 - 谷氨酰胺溶液(Life Technologies,Gibco ,目录号:10378-016)
  4. 2-巯基乙醇(Life Technologies,Invitrogen TM,目录号:21985-023)
  5. 佛波醇12-豆蔻酸酯-13-乙酸酯(PMA)(Sigma-Aldrich,目录号:79346)
  6. 离子霉素(Sigma-Aldrich,目录号:19657)
  7. 5(和-6) - 羧基荧光素二乙酸酯,琥珀酰亚胺酯(5(6)-CFDA,SE)(CFSE)(Life Technologies,Molecular Probes ,目录号:C1157)
  8. 7-羟基-9H-(1,3-二氯-9,9-二甲基吖啶-2-酮(DDAO)(Life Technologies,Invitrogen TM,目录号:C34553)
  9. 5-(和-6) - (((4-氯甲基)苯甲酰基)氨基)四甲基罗丹明(CMTMR)(Life Technologies,Invitrogen TM,目录号:C2927)
  10. 16%多聚甲醛溶液(PFA)(16%甲醛,EM级)(Electron Microscopy Sciences,目录号:15710)
  11. Vectashield Hardset with Dapi(VWR International,目录号:101098-050)
  12. 在H 2 O中的0.1%(w/v)的聚-L-赖氨酸溶液(Sigma-Aldrich,目录号:P8920)
  13. SL8(卵白蛋白(257-264))(AnaSpec,目录号:60193)
  14. PBS(Life Technologies,目录号:14190-44)
  15. 媒体(见配方)
  16. 2%PFA(参见配方)
  17. 聚-L-赖氨酸涂层室(见配方)


  1. 离心机
  2. 流式细胞仪
  3. 荧光显微镜
  4. 37℃,5%CO 2细胞培养箱
  5. 24孔板(Corning,Costar ,目录号:3524)
  6. 40μm细胞过滤器(BD Biosciences,Falcon ,目录号:352340)
  7. FACS管,聚苯乙烯(BD Biosciences,Falcon ,目录号:352054)
  8. 8孔腔室载玻片(Thermo Fisher Scientific,Lab-Tek TM,目录号:177445)


  1. T-APC共轭测定
    1. 该方案使用来自未免疫小鼠的纯化的OT-I CD8 + T细胞,其是 对于识别鸡卵OVA来源的SIINFEKL的TCR是转基因的 肽(SL8)在MHC I类分子H2-k抗体的上下文中。一样  方案可以与对其他抗原特异的T细胞一起使用。 BMDC (骨髓来源的树突状细胞)用作APC,但DC来自 其他来源或许多细胞系,可用作APC。
    2. 一旦细胞被标记,它们应该尽可能地避免光。

    1. 细胞标记:以10×10 6个细胞/ml(通常在2ml中为20×10 6个细胞)的浓度在PBS中重悬悬BMDC和纯化的初始OT-I细胞, 。在BMDCs上加入终浓度为4μM的DDAO,在OT-I细胞上加入终浓度为1μM的CFSE。在37℃,5%CO 2孵育30分钟 注意:CFSE和DDAO可以替代其他染料。使用这两种染料的优点是需要在流式细胞仪上几乎没有补偿。
    2. 通过在室温下以300xg离心5分钟,用5体积的培养基(如果开始于20×10 6个细胞,则为10ml)洗涤细胞三次。 />
    3. 脉冲APC:以10×10 6个细胞/ml的浓度将BMDC重悬在培养基中。加入SL8,终浓度范围为0-1,000ng/ml(通常使用5种不同浓度),并在37℃,5%CO 2下孵育60分钟。
    4. 虽然APC是抗原脉冲,将OT-I细胞以4×10 6个细胞/ml重悬于培养基(通常为5ml中的20×10 6个细胞)中。每个FACS管添加50微升OT-I细胞。离开细胞在37℃5%CO 2,直到APC准备好。
    5. 通过在室温下以300xg离心5分钟,用5体积的培养基洗涤BMDC三次。
    6. 在培养基中以4×10 6个细胞/ml重悬BMDC
    7. 向每个含有OT-I细胞的FACS管中加入50μl的BMDCs。立即以228×g(1,000rpm)在20℃下旋转1分钟。立即置于37℃。
    8. 固定细胞:一旦所需的时间量完成,加入100μl每个样品的温暖(20°C)2%PFA。 非常轻轻地混合,通过上下吹吸或快速涡旋低。 在室温(约20℃)孵育10分钟。
    9. 在流式细胞仪上运行。 基于双重CFSE/DDAO阳性评估偶联的百分比(图1)。

      图1.T-APC偶联测定 - 实施例。 CFSE标记 OT-I细胞在37℃下与DDAO标记的BMDC一起孵育30分钟   其用0(左图)或100ng/ml(右图)SL8脉冲 肽。 首先使用SSC/FSC门(上部)排除细胞碎片 面板)。 耦合单元是DDAO + 和CFSE +

  2. T-T聚类测定
    1. 在该方案中,T细胞用PMA和离子霉素活化 旁路对APC的需要和每个T细胞可以被激活 办法。 然而,相同的协议可以用于与激活的T细胞 APC,抗CD3和抗CD28等...
    2. 作为示例,该协议比较OT-1细胞的容量 缺乏整合素ICAM-1形成同型相互作用 与对照OT-I细胞相比。 但是可以使用相同的协议 调查T细胞聚集,阻断抗体等的动力学。

    1. 细胞标记:以10×10 6个细胞/ml(通常是2ml中的20×10 6个细胞)的浓度将纯化的OT-I细胞重悬在PBS中。在对照细胞上加入终浓度为1μM的CFSE,在ICAM-1 -/- 细胞上加入终浓度为4μM的CMTMR。在37℃,5%CO 2孵育30分钟
    2. 通过在室温下以300×g离心5分钟,用5体积的培养基洗涤细胞三次。
    3. T细胞活化:以2×10 6个细胞/ml的浓度在培养基中重悬OT-1细胞。 WT和ICAM-1 -/- 细胞以比率1混合,然后通过在培养基中直接加入5ng/ml PMA和50ng/ml离子霉素来活化细胞。在24孔板的每孔中铺板2ml细胞悬浮液
    4. 活化后16至24小时,通过流式细胞术(步骤B5)或显微镜(步骤B6)分析聚类中的细胞百分比。
    5. 通过流式细胞术分析:通过在20℃下加入相同体积的2%PFA 10分钟来固定细胞。固定的细胞通过40μm过滤器进入FACS管。经过的细胞代表"非聚集"部分。保留在过滤器上的细胞用1ml PBS 2mM EDTA(以破坏细胞簇)洗涤并代表"聚集的"级分。两种级分在流式细胞仪上运行(图2)

      图2.T-T聚类分析 - 通过流式细胞术分析。 将CFSE标记的WT OT-I细胞和CMTMR标记的ICAM-1-OT-I细胞混合并用PMA和离子霉素刺激。活化后24 h,聚集的细胞 通过40μm过滤器与非聚集的细胞分离,并通过流式细胞术分析两种级分。首先使用SSC/FSC门排除细胞碎片(上图)。聚集的细胞 - 左图;非聚簇细胞 - 右图。

    6. 通过显微镜分析:将细胞(通常300μl的细胞悬浮液)转移到聚-L-赖氨酸包被的室上,并在37℃,5%CO 2下孵育10分钟。然后小心地除去培养基并用200μl的2%PFA替换。 10分钟后,小心地除去PFA,并用200μlPBS代替。取出PBS,让其干燥2分钟。取下腔室垫圈,并用一滴Vectashield在盖玻片上安装。用epifluorescent显微镜分析 注意:切割移液管吸头的尖端,以确保在将细胞悬浮液应用于培养室时,细胞群不会被移液管破坏。


  1. 媒体(555毫升)
    500ml RPMI Medium 1640
    500μl50mMβ-巯基乙醇 5ml青霉素 - 链霉素 - 谷氨酰胺溶液 保持在4°C
  2. 2%PFA
    稀释1ml的16%PFA溶液在7ml PBS中
  3. 聚-L-赖氨酸涂层室
    稀释聚-L-赖氨酸储备溶液1/100 in dH 2 sub *
    用dH 2 O彻底清洗,并让室干燥至少2小时


该协议改编自以前发表的论文Gerard等人(2013)。 该方案在Matthew F.Krummel的实验室中改编,并且得自幼年糖尿病基金会(MFK)和NIH R01AI52116(MFK)的赠款。


  1. Gérard,A.,Khan,O.,Beemiller,P.,Oswald,E.,Hu,J.,Matloubian,M.and Krummel,M.F。 次级T细胞-T细胞突触相互作用驱动保护性CD8 + T细胞的分化。 Nat Immunol 14(4):356-363
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Gérard, A. (2013). In vitro T Cell–DC and T Cell–T Cell Clustering Assays. Bio-protocol 3(20): e933. DOI: 10.21769/BioProtoc.933.