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Calcium Mobilisation Assay in Response to Chemokine Stimulation
趋化因子刺激下淋巴细胞内钙动员能力的分析   

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Abstract

This assay is used to measure calcium mobilization in lymphocytes (either in primary cells or cell lines) in response to chemokine stimulation using ratiometric analysis. It has also been used for measuring TCR mediated calcium flux. In addition, the same labeling procedure with the addition of brilliant black (a quenching agent) (Sigma-Aldrich, catalog number: 211842) in the loading buffer (at 100 μM) allows for quantification using the FLIPR system on poly-D-lysine plates. Probenicid is an anion-exchange protein inhibitor and prevents the extrusion of the dyes by organic ion transporters.

Materials and Reagents

  1. Cells of choice
  2. RPMI 1640 media (Life Technologies, Invitrogen™, catalog number: 11875-119 )
  3. Fetal bovine serum (FBS) (Life Technologies, Invitrogen™, catalog number: 10437-028 )
  4. PTX (List Biological Labs, catalog number: 183 )
  5. HBSS (Life Technologies, Invitrogen™, catalog number: 14185-052 )
  6. BSA (Sigma Aldrich, catalog number: A7030 )
  7. HEPES (Life Technologies, Invitrogen™, catalog number: 15630-080 )
  8. Probenecid (Sigma Aldrich, catalog number: P8761 )
  9. Fluo-4 (Life Technologies, Invitrogen™, catalog number: F14201 )
  10. Fura-red (Life Technologies, Invitrogen™, catalog number: F3021 )
  11. Pluronic F-127 (Life Technologies, Invitrogen™, catalog number: P-3000MP )
  12. DMSO (Sigma Aldrich, catalog number: D2650 )
  13. Ionomycin (Sigma Aldrich, catalog number: I0634 )
  14. NaOH (Thermo Fisher Scientific, catalog number: BP359-212 )
  15. Assay buffer (see Recipes)
  16. Loading buffer (see Recipes)

Equipment

  1. FCM (FACS LSR II machine)
  2. FACS tube

Procedure

  1. Cells are collected, washed and resuspended at ~1-2 x 106 cells/ml in RPMI 1640 media (no FBS).
  2. If treated with PTX, then samples are split in 2 with one receiving 100 ng/ml PTX and the other PBS. Tubes are rotated at 37 °C for 3-4 h.
  3. If cells are not pretreated with PTX, then cells are rotated for 1 h at 37 °C.
  4. Cells are then washed twice with assay buffer+ 2.5 mM probenecid.
  5. Resuspend cells in loading buffer (assay buffer + probenecid + 2.28 µg/ml Fluo-4 + 0.91 µg/ml Fura-red) at ~4 x 106 cells/ml. N.B. In both cases, 50 µg of Fluo-4 and Fura-red are dissolved in 22 µl pluronic F-127 (20% solution in DMSO) and 22 µl DMSO.
  6. Light protected tubes are rotated for 30 min at 37 °C.
  7. Cells are then washed once and resuspended with assay buffer + probenecid at ~ 5 x 106 cells/ml.
  8. For SDF-mediated calcium flux analysis, 450 µl of cells are aliquotted into a FACS tube and basal ratio of FITC/PerCP-Cy5.5 levels are recorded. At 1 min, cells are stimulated with 50 µl 10 µg/ml (~125 nM final) SDF-1 and responses recorded for a further 3 min. Finally, cells are stimulated with 50 µl 20 µM ionomycin.
    Note: SDF-1 can be replaced by other chemokines as you desire. However, optimal dose needs to be determined.

Notes

  1. Assay buffer can be stored at 4 °C. Allow to reach room temperature before use.
  2. Probenecid is prepared fresh each time in 1 N NaOH (stock = 500 mM).
  3. Unused Fluo-4 and Fura-Red, once reconstituted in DMSO/Pluronic acid, can be refrozen once for use on a future date.

Recipes

  1. Assay buffer (pH 7.4)
    HBSS
    0.1% BSA
    HEPES (pH 7.4)    25 mM
    Probenecid            2.5 mM
  2. Loading buffer
    Assay buffer
    Probenecid
    2.28 µg/ml Fluo-4
    0.91 µg/ml Fura-red

Acknowledgments

This protocol was adapted from previously published reports (Cronshaw et al., 2006 and Cronshaw et al., 2010).

References

  1. Cronshaw, D. G., Kouroumalis, A., Parry, R., Webb, A., Brown, Z. and Ward, S. G. (2006). Evidence that phospholipase-C-dependent, calcium-independent mechanisms are required for directional migration of T-lymphocytes in response to the CCR4 ligands CCL17 and CCL22. J Leukoc Biol 79(6): 1369-1380.
  2. Cronshaw, D. G., Nie, Y., Waite, J. and Zou, Y. R. (2010). An essential role of the cytoplasmic tail of CXCR4 in G-protein signaling and organogenesis. PLoS One 5(11): e15397.

简介

该测定用于使用比率分析测量响应趋化因子刺激的淋巴细胞(原代细胞或细胞系)中的钙动员。 它也被用于测量TCR介导的钙通量。 此外,在加样缓冲液(100μM)中加入亮黑(淬灭剂)(Sigma-Aldrich,目录号:211842)的相同标记方法允许使用FLIPR系统定量聚D-赖氨酸 板。 丙磺舒是阴离子交换蛋白抑制剂,并通过有机离子转运蛋白防止染料的挤出。

材料和试剂

  1. 首选单元
  2. RPMI 1640培养基(Life Technologies,Invitrogen TM,目录号:11875-119)
  3. 胎牛血清(FBS)(Life Technologies,Invitrogen TM,目录号:10437-028)
  4. PTX(List Biological Labs,目录号:183)
  5. HBSS(Life Technologies,Invitrogen TM,目录号:14185-052)
  6. BSA(Sigma Aldrich,目录号:A7030)
  7. HEPES(Life Technologies,Invitrogen TM,目录号:15630-080)
  8. 丙磺舒(Sigma Aldrich,目录号:P8761)
  9. Fluo-4(Life Technologies,Invitrogen TM,目录号:F14201)
  10. Fura-red(Life Technologies,Invitrogen TM,目录号:F3021)
  11. Pluronic F-127(Life Technologies,Invitrogen TM,目录号:P-3000MP)
  12. DMSO(Sigma Aldrich,目录号:D2650)
  13. 离子霉素(Sigma Aldrich,目录号:I0634)
  14. NaOH(Thermo Fisher Scientific,目录号:BP359-212)
  15. 测试缓冲区(参见配方)
  16. 加载缓冲区(参见配方)

设备

  1. FCM(FACS LSR II机器)
  2. FACS管

程序

  1. 收集细胞,洗涤并以约1-2×10 6个细胞/ml重悬于RPMI 1640培养基(无FBS)中。
  2. 如果用PTX处理,则将样品分成2份,一份接受100ng/ml PTX,另一份接受另一份PBS。 将管在37℃下旋转3-4小时。
  3. 如果细胞不用PTX预处理,则细胞在37℃下旋转1小时
  4. 然后用测定缓冲液+ 2.5mM丙磺舒洗涤细胞两次
  5. 以〜4×10 6个细胞/ml重悬细胞于上样缓冲液(测定缓冲液+丙磺舒+2.28μg/ml Fluo-4 +0.91μg/ml Fura-red) N.B.在这两种情况下,将50μgFluo-4和Fura-red溶解于22μl普流罗尼F-127(20%的DMSO溶液)和22μlDMSO中。
  6. 将受保护的管在37℃下旋转30分钟
  7. 然后将细胞洗涤一次,并用测定缓冲液+丙磺舒以约5×10 6个细胞/ml重悬。
  8. 对于SDF介导的钙流量分析,将450μl细胞等分到FACS管中,并记录FITC/PerCP-Cy5.5水平的基础比率。在1分钟时,用50μl10μg/ml(〜125nM最终)SDF-1刺激细胞,并记录反应另外3分钟。最后,用50μl20μM离子霉素刺激细胞。
    注意:SDF-1可以根据需要由其他趋化因子替代。但是,需要确定最佳剂量。

笔记

  1. 测定缓冲液可以在4°C储存。 使用前应达到室温。
  2. 每次在1N NaOH(储备液= 500mM)中新鲜制备丙磺舒。
  3. 未使用的Fluo-4和Fura-Red,一旦在DMSO/Pluronic酸中重构,可以再冷冻一次,以备将来使用。

食谱

  1. 测定缓冲液(pH 7.4)
    HBSS
    0.1%BSA
    HEPES(pH 7.4)    25 mM
    Probenecid             2.5mM
  2. 加载缓冲区
    测定缓冲区
    泛素
    2.28μg/ml Fluo-4
    0.91μg/ml Fura-red

致谢

该方案改编自以前公开的报道(Cronshaw等人,2006和Cronshaw等人,2010)。

参考文献

  1. Cronshaw,D.G.,Kouroumalis,A.,Parry,R.,Webb,A.,Brown,Z.and Ward,S.G。(2006)。 有证据表明磷脂酶C依赖性,钙依赖性机制是T淋巴细胞定向迁移所必需的 响应于CCR4配体CCL17和CCL22。 Leukoc Biol 79(6):1369-1380。
  2. Cronshaw,D.G.,Nie,Y.,Waite,J。和Zou,Y.R。(2010)。 CXCR4的胞质尾部在G蛋白信号传导和器官形成中的重要作用。 PLoS One 5(11):e15397。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Cronshaw, D. G. (2012). Calcium Mobilisation Assay in Response to Chemokine Stimulation. Bio-protocol 2(9): e160. DOI: 10.21769/BioProtoc.160.
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Hi,
Thank you very much for the protocol. Once acquired, how do you analyse it on Flowjo. Is there a protocol for that?
Best,
Yasar
9/20/2012 4:13:12 PM Reply