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Flow Cytometric Analysis of Calcium Influx Assay in T cells
利用流式细胞术分析T淋巴细胞中钙流变化   

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Abstract

Calcium influx is one of the key signaling events upon stimulation of T cell receptors (TCR) and plays an important role for T cell activation, proliferation, and differentiation. Phorbol myristate acetate (PMA) and calcium ionophore ionomycin are commonly utilized as stimulants in a variety of immunologic assays including T cell activation. PMA is a protein kinase C (PKC) activator, resulting in the activation of Ras, a small GPTase. When PMA and ionomycin are used together, TCR signaling downstream of PKC and Ras can be activated without activation of TCR-triggerd signaling events. This protocol describes the flow cytometry analysis of intracellular calcium influx in T cells stimulated with PMA and ionomycin.

Materials and Reagents

  1. Jurkat T cells (ATCC, catalog number: TIB-152 )
  2. DMSO (Sigma-Aldrich, catalog number: 472301 )  
  3. RPMI media 1640 (Life Technologies, Gibco®, catalog number: 21875-034 )
  4. Fetal Bovine Serum (FBS) (Life Technologies, Gibco®, catalog number: 16000044 )
  5. Penicillin/streptomycin (pen/strep) (Life Technologies, Gibco®, catalog number: 15140-122 )
  6. PMA (Sigma-Aldrich, catalog number: P1585 ) (Store at -20 °C)
  7. Ionomycin (Sigma-Aldrich, catalog number: I0623 ) (Store at -20 °C)
  8. Calcium Assay Kit (BD Biosciences, catalog number: 640176 )
  9. Complete RPMI media (see Recipes)
  10. PMA stock solution (see Recipes)
  11. Ionomycin stock solution (see Recipes)

Equipment

  1. Centrifuge (Eppendorf, model: 5810R )
  2. 37 °C 5% CO2 Cell culture incubator
  3. Cell Counter
  4. BD CantoII FACS machine
  5. 5 ml round-bottom FACS tube (BD Biosciences, catalog number: 352003 )

Software

  1. FACS DIVA software
  2. FlowJo software

Procedure

  1. Preparation (For 4 samples)
    1. Preparation of Jurkat T cells:
      1. Count cells growing exponentially (1 x 106 per assay).
      2. Wash cells once with pre-warmed complete RPMI media at 200 x g for 5 min.
      3. Place 1 x 106 cells/250 μl of fresh complete RPMI media into a 5 ml FACS tube.
    2. Preparation of 1x enhancing solution (from Calcium Assay Kit) (2 ml for 4 assays):
      1. Mix 200 μl of 10x enhancing solution with 1.8 ml of assay buffer.
      2. Keep it at room temperature.
    3. Preparation of indicator (from Calcium Assay Kit):
      1. Equilibrate a vial of indicator (stored at -20 °C) at room temperature for 5 min.
      2. Add 100 μl of 100% DMSO.
      3. Mix well by pipetting up and down multiple times.
      4. Store at RT for 10 min to stabilize completely.
      5. Store unused indicator in a small aliquot at -20 °C until use.
    4. Preparation of 1x loading dye:
      Mix 2 ml of 1x enhancing solution with 2 μl of indicator prepared above.
    5. Loading dye to cells:
      Add 250 μl of 1x loading dye (prepared in step 4) into each tube containing cells.
    6. Incubate tube for 1 h in 37 °C CO2 Incubator.
    7. Cool down tube at RT for 20 min before analysis

  2. FACS analysis
    1. Open the FACS DIVA software.
    2. Draw a dot plot (Time is on the X-axis, and FITC is on the Y-axis).
    3. Place a tube to the FACS machine.
    4. Click “Record Data” for 1 min to obtain the basal level of signal.
    5. Click “Stop Acquiring” and remove the tube from the FACS machine.
    6. Immediately add 1 μl of stimulator [a mixture of PMA (50 ng/ml) and ionomycin (1 μg/ml)].
    7. Vortex briefly and place the tube back to the FACS machine.
    8. Click “Record Data” for additional 3 min.
    9. Click “Append” to attach acquired signal to the 1 min basal level of signal acquired in step 4.
    10. Click “Stop Acquiring” to finish the assay.
    11. Analyze each data with FlowJo software by choosing kinetic mode.
    12. Overlay sample data on the control data to display the difference of signal between the control and sample on one Figure as shown in Figure 1.


      Figure 1. Intracellular Ca2+ influx in IKKγ/NEMO-deficient Jurkat T cells stably complemented with Ha-tagged IKKγ/NEMO WT or mutants (S377E, S377A, Y374D, Y374F). Cells were loaded with Ca2+ indicator for 1 h at 37 °C. Intracellular Ca2+ influx upon PMA and ionomycin (P + I) treatment was monitored for 5 min by flow cytometry analysis. Vec stands for vector control.

Recipes

  1. Complete RPMI media
    RPMI containing 10% FBS and 1% penicillin/streptomycin
  2. PMA stock solution
    50 ng/ml DMSO
  3. Ionomycin stock solution
    1 μg/ml DMSO

Acknowledgments

This protocol is adapted from Lee et al. (2012).

References

  1. Lee, S. H., Toth, Z., Wong, L. Y., Brulois, K., Nguyen, J., Lee, J. Y., Zandi, E. and Jung, J. U. (2012). Novel Phosphorylations of IKKγ/NEMO. MBio 3(6): e00411-00412.

简介

钙流入是刺激T细胞受体(TCR)的关键信号事件之一,并且对T细胞活化,增殖和分化起重要作用。 佛波醇肉豆蔻酸乙酸酯(PMA)和钙离子载体离子霉素通常在多种免疫测定(包括T细胞活化)中用作刺激剂。 PMA是蛋白激酶C(PKC)激活剂,导致Ras(一种小GPTase)的活化。 当PMA和离子霉素一起使用时,PKC和Ras下游的TCR信号传导可以在没有激活TCR-触发信号事件的情况下被激活。 该协议描述了用PMA和离子霉素刺激的T细胞中的细胞内钙流入的流式细胞术分析。

材料和试剂

  1. Jurkat T细胞(ATCC,目录号:TIB-152)
  2. DMSO(Sigma-Aldrich,目录号:472301)
  3. RPMI培养基1640(Life Technologies,Gibco ,目录号:21875-034)
  4. 胎牛血清(FBS)(Life Technologies,Gibco ,目录号:16000044)
  5. 青霉素/链霉素(pen/strep)(Life Technologies,Gibco ,目录号:15140-122)
  6. PMA(Sigma-Aldrich,目录号:P1585)(-20℃保存)
  7. 离子霉素(Sigma-Aldrich,目录号:I0623)(-20℃保存)
  8. 钙测定试剂盒(BD Biosciences,目录号:640176)
  9. 完成RPMI媒体(参见配方)
  10. PMA储备溶液(见配方)
  11. 离子霉素储备溶液(见配方)

设备

  1. 离心机(Eppendorf,型号:5810R)
  2. 37℃5%CO 2细胞培养箱
  3. 单元格计数器
  4. BD CantoII FACS机器
  5. 5ml圆底FACS管(BD Biosciences,目录号:352003)

软件

  1. FACS DIVA软件
  2. FlowJo软件

程序

  1. 制备(对于4个样品)
    1. Jurkat T细胞的制备:
      1. 计数细胞指数生长(每次测定1×10 6次)
      2. 用预热的完全RPMI培养基在200×g下洗涤细胞5分钟。
      3. 将1×10 6个细胞/250μl新鲜完全RPMI培养基置于5ml FACS管中。
    2. 1x增强溶液(来自钙测定试剂盒)(2ml,用于4次测定)的制备:
      1. 混合200μl的10倍增强溶液与1.8毫升测定缓冲液
      2. 保持室温。
    3. 指示剂的制备(来自钙测定试剂盒):
      1. 平衡指示器(储存在-20℃)在室温下5分钟。
      2. 加入100μl的100%DMSO。
      3. 通过多次上下吹吸混匀。
      4. 在室温储存10分钟以完全稳定。
      5. 将未使用的指示物储存在-20℃的小份中直至使用。
    4. 1×装载染料的制备:
      混合2毫升1x增强溶液与2微升上面制备的指标。
    5. 加载染料到细胞:
      加入250微升1x加载染料(在步骤4中制备)到每个含有细胞的管中。
    6. 在37℃CO 2孵育器中孵育管1小时。
    7. 在室温下冷却管20分钟,然后分析

  2. FACS分析
    1. 打开FACS DIVA软件。
    2. 绘制点图(时间在X轴,FITC在Y轴)。
    3. 将管插入FACS机器。
    4. 单击"记录数据"1分钟以获得基础水平的信号
    5. 单击"停止获取"并从FACS机器中取出试管。
    6. 立即加入1μl刺激剂[PMA(50ng/ml)和离子霉素(1μg/ml)的混合物]。
    7. 短暂涡旋并将管放回FACS机器。
    8. 再点击"记录数据"3分钟。
    9. 单击"附加"将获取的信号附加到步骤4中获取的信号的1分钟基础水平。
    10. 单击"停止采集"完成测定。
    11. 通过选择动力学模式,用FlowJo软件分析每个数据。
    12. 将控制数据上的样本数据重叠显示,如图1所示,在一个图上显示控件和样本之间的信号差异。


      图1.细胞内Ca 2 + 在IKKγ/NEMO缺陷型Jurkat T细胞中的流入与Ha标记的IKKγ/NEMO WT或突变体(S377E,S377A,Y374D,Y374F)。在37℃下用Ca 2+指示剂加载细胞1小时。 通过流式细胞术分析监测在PMA和离子霉素(P + I)处理上的细胞内Ca 2+流入5分钟。 Vec代表矢量控制。

食谱

  1. 完成RPMI媒体
    含有10%FBS和1%青霉素/链霉素的RPMI
  2. PMA储备液
    50ng/ml DMSO
  3. 离子霉素储备液
    1μg/ml DMSO

致谢

该协议改编自Lee等人(2012)。

参考文献

  1. Lee,S.H.,Toth,Z.,Wong,L.Y.,Brulois,K.,Nguyen,J.,Lee,J.Y.,Zandi,E.and Jung,J.U。 IKKγ/NEMO的新型磷酸化 MBio 3( 6):e00411-00412。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Lee, S. (2013). Flow Cytometric Analysis of Calcium Influx Assay in T cells. Bio-protocol 3(18): e910. DOI: 10.21769/BioProtoc.910.
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