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T cell Calcium Mobilization Study (Flow Cytometry)
T细胞钙离子流动性分析(流式细胞仪)   

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Abstract

Antigen recognition and activation of T cell receptor (TCR) triggers transient calcium release from intracellular compartments and subsequent sustained calcium influx through cell surface Icrac channels. Sustained elevation of the cytoplasmic calcium level activates many calcium-dependent enzymes and transcription factors, which are essential for T cell activation and function. This protocol uses non-ratiometric dyes, in combination with flow cytometry, to monitor TCR-triggered calcium changes over time, and is a simple assay to examine the existence of T cell calcium mobilization defects in transgenic mice.

Materials and Reagents

  1. Anti-CD3 Armernian hamster primary antibody (BD Biosciences, catalog number: 553057 , NA/LE)
  2. Goat anti-Armernian Hamster IgG antibody (Jackson ImmunoResearch, catalog number: 127-005-099 )
  3. Phosphate buffered saline (PBS)
  4. CaCl2
  5. MgCl2
  6. DMSO (Merck KGaA, Calbiochem®, catalog number: 540025 )
  7. Fluo-3 AM in DMSO (Life Technologies, Molecular Probes®, catalog number: F-1242 )
    (Note: Fluo-3 fluorescence is calcium-dependent) (see Recipes)
  8. Fura Red AM in DMSO (Life Technologies, Molecular Probes®, catalog number: F-3020 ) (Note: Fura Red fluorescence is calcium-independent. Fura Red serves as a control of dye loading efficiency) (see Recipes)
  9. Pluronic F-127 in DMSO (Life Technologies, Molecular Probes®, catalog number: F-1242) (see Recipes)
  10. Hanks buffered solution (HBSS) (Life Technologies, Gibco®, catalog number: 14170-112 ) (see Recipes)
  11. Dye loading buffer (see Recipes)

Equipment

  1. Flow Cytometry

Procedure

  1. Prepare dye loading buffer 2 ml for one sample.
  2. Suspend cells at 5 x 106 cells/ml in 1 ml dye loading buffer and incubate 30 min at 37 °C.
  3. Spin down cells 5 min at 1,000 rpm.
  4. Stain cells with 5 μg/ml anti-CD3 hamster primary antibody (no azide) for 30 min on ice or at 4 °C (0.5 μg/100 μl PBS).
  5. Wash cells once.
  6. Resuspend cells in 3 ml HBSS/Ca/Mg/FBS at 3 x 106 cells/ml and store at RT and protect from light.
  7. For calcium mobilization, warm up samples at 37 °C for 5-10 min. Submit samples to flow cytometry for calcium baseline measurement. After 5 min, add 5 μg/ml goat anti-hamster IgG antibody (15 μg/3 ml), mix well and immediately continue the measurement with flow cytometry. To maintain the incubation temperature, a small beaker containing water prewarmed to 37 °C is necessary to bath sample tubes during the time course of measurement.

Recipes

  1. Dye loading buffer (2 ml for one sample)
    1. HBSS/Ca/Mg/FBS
      HBSS
      20 ml
      1 mM CaCl2
      26 μl
      1 mM MgCl2
      22 μl
      FBS
      200 μl
    2. Dye loading buffer
      HBSS/Ca/Mg/FBS
      2 ml
      10% pluronic F-127
      4 μl
      4 mg/ml Fluo-3 AM
      2 μl
      10 mg/ml Fura Red AM
      2 μl

  2. Hanks buffered solution (HBSS) supplemented with 1.3 mM CaCl2 and 1.1 mM MgCl2.
  3. 10% (w/v) pluronic F-127 in DMSO, 10%, store at RT.
  4. 4 mg/ml Fluo-3 AM in DMSO (20 x 50 μg) aliquot and store in a -20 °C dessicator.
  5. 10 mg/ml Fura Red AM in DMSO (500 μg) aliquot and store in a -20 °C dessicator.

Acknowledgments

This protocol was previously used in and adapted from Huang et al. (2008).

References

  1. Huang, G. N., Huso, D. L., Bouyain, S., Tu, J., McCorkell, K. A., May, M. J., Zhu, Y., Lutz, M., Collins, S., Dehoff, M., Kang, S., Whartenby, K., Powell, J., Leahy, D. and Worley, P. F. (2008). NFAT binding and regulation of T cell activation by the cytoplasmic scaffolding Homer proteins. Science 319(5862): 476-481.

简介

抗原识别和T细胞受体(TCR)的激活触发从细胞内区室瞬时钙释放和随后通过细胞表面Icrac通道的持续钙流入。 细胞质钙水平的持续升高激活许多钙依赖性酶和转录因子,其对于T细胞活化和功能是必需的。 该协议使用非比率染料,结合流式细胞术,监测TCR触发的钙随时间的变化,是一个简单的测定,检查转基因小鼠的T细胞钙动员缺陷的存在。

材料和试剂

  1. 抗CD3 Armernian仓鼠一抗(BD Biosciences,目录号:553057,NA/LE)
  2. 山羊抗阿默生仓鼠IgG抗体(Jackson ImmunoResearch,目录号:127-005-099)
  3. 磷酸盐缓冲盐水(PBS)
  4. CaCl <2>
  5. MgCl 2
  6. DMSO(Merck KGaA,Calbiochem ,目录号:540025)
  7. DMSO中的Fluo-3AM(Life Technologies,Molecular Probes ,目录号:F-1242)
    (注意:Fluo-3荧光是钙依赖的)(参见配方)
  8. Fura Red AM在DMSO中(Life Technologies,Molecular Probes ,目录号:F-3020)(注意:Fura Red荧光是钙非依赖性的,Fura Red用作染料加载的对照 效率)(参见配方)
  9. 在DMSO中的Pluronic F-127(Life Technologies,Molecular Probes ,目录号:F-1242)(参见配方)
  10. Hanks缓冲溶液(HBSS)(Life Technologies,Gibco ,目录号:14170-112)(参见Recipes)
  11. 染料加载缓冲液(参见配方)

设备

  1. 流式细胞术

程序

  1. 准备一个样品的染料加样缓冲液2ml
  2. 将细胞以5×10 6个细胞/ml悬浮于1ml染色上样缓冲液中,并在37℃下孵育30分钟。
  3. 以1,000 rpm速度旋转细胞5分钟。
  4. 在冰上或在4℃下用5μg/ml抗CD3仓鼠一抗(无叠氮化物)染色细胞30分钟(0.5μg/100μlPBS)。
  5. 洗涤细胞一次。
  6. 在3ml HBSS/Ca/Mg/FBS中以3×10 6个细胞/ml重悬细胞,并在室温下保存并避光。
  7. 对于钙动员,将样品在37℃预热5-10分钟。 提交样品到流式细胞仪进行钙基线测量。 5分钟后,加入5μg/ml山羊抗仓鼠IgG抗体(15μg/3ml),充分混合,立即用流式细胞仪继续测量。 为了保持培养温度,在测量的时间过程中,需要一个装有预热到37℃的水的小烧杯来洗涤样品管。

食谱

  1. 染料加样缓冲液(对于一个样品为2ml)
    1. HBSS/Ca/Mg/FBS
      HBSS
      20ml
      1mM CaCl 2
      26微升
      1mM MgCl 2
      22微升
      FBS
      200μl
    2. 染料加载缓冲液
      HBSS/Ca/Mg/FBS
      2 ml
      10%普流尼克F-127 4微升
      4 mg/ml Fluo-3 AM
      2微升
      10mg/ml Fura Red AM
      2微升

  2. 补充有1.3mM CaCl 2和1.1mM MgCl 2的Hanks缓冲溶液(HBSS)。
  3. 10%(w/v)pluronic F-127的DMSO溶液,10%,室温贮存
  4. 4 mg/ml Fluo-3 AM的DMSO溶液(20 x 50μg),保存在-20°C的干燥器中。
  5. 10mg/ml的DMSO(500μg)中的Fura Red AM等分试样,并储存在-20℃的干燥器中。

致谢

该方案以前用于和改编自Huang等人(2008)。

参考文献

  1. Huang,GN,Huso,DL,Bouyain,S.,Tu,J.,McCorkell,KA,May,MJ,Zhu,Y.,Lutz,M.,Collins,S.,Dehoff,M.,Kang, ,Whartenby,K.,Powell,J.,Leahy,D。和Worley,PF(2008)。 NFAT通过胞质支架Homer蛋白结合和调节T细胞活化。 Science 319(5862):476-481。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Huang, G. N. (2012). T cell Calcium Mobilization Study (Flow Cytometry). Bio-protocol 2(9): e171. DOI: 10.21769/BioProtoc.171.
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