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FACS Staining for Follicular Helper T Cells
滤泡性辅助性T细胞的流式细胞分析

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Abstract

Germinal center is the primary site for B cells to undergo somatic hypermutation and class switching. A recently discovered subset of T cells known as T follicular helper (TFH) cells are essential for germinal center reaction and thus have been the subjects of intensive study in the recent years. This protocol describes a reliable way to stain this population for flow analysis. This protocol was developed or modified in Dr. Anne Davidson’s lab at Feinstein Institute for Medical Research.

Keywords: Flow cytometry(流式细胞仪), Mouse(小鼠), Follicular helper T cells(滤泡辅助性T细胞), Germinal center(生发中心)

Materials and Reagents

  1. Antibodies
    1. Rat anti-mouse CXCR5-biotin (BD Biosciences, Pharmingen™, catalog number: 551960 )
    2. Goat anti-rat IgG-biotin (BD Biosciences, Pharmingen™, catalog number: 554014 )
    3. Straptavidin-PE Cy7 (BD Biosciences, Pharmingen™, catalog number: 557598 )
    4. Rat anti-mouse B220-pacific blue 450 (eBioscience,  catalog number: 48-0452-82 )
    5. Rat anti-mouse CD11b-pacific blue 450 (eBioscience,  catalog number: 48-0112-82 )
    6. Rat anti-mouse CD4-APC eFluor 780 (eBioscience, catalog number: 47-0042-82 )
    7. Hamster anti-mouse PD-1-PE (eBioscience, catalog number: 12-9985-82 )
    8. Mouse Fc block (BD Biosciences, Pharmingen™, catalog number: 553141 )

  2. Other materials
    1. Single cell suspensions derived from murine spleen samples
    2. Fetal bovine serum (FBS) (Hyclone)
    3. Dulbecco’s modification eagle medium (DMEM) (Life Technologies, Invitrogen™)
    4. Ammonium chloride (0.17 M, filtered, autoclaved) (pH7.4)
    5. DAPI nucleic acid stain (Life Technologies, Invitrogen™)
    6. FACS buffer (see Recipes)

Equipment

  1. BD LSR II flow cytometer
  2. Conical tubes (Corning)

Procedure

  1. Harvest the spleen and create single cell suspension by gently smashing spleen pieces with the frosted surface of a pair of microscope slides in 5 ml of DMEM.
  2. Transfer the cells into 50 ml conical tubes and spin down the cells at 300 RCF for 5 min at 4 °C.
  3. Discard the supernatant with aspiration without disturbing the pellet.
  4. Resuspend the cells with 5 ml of 0.17 M ammonium chloride and keep the cells on ice for 5 min.
  5. Add 15 ml DMEM to the cells and spin at 300 RCF for 5 min at 4 °C.
  6. Discard the supernatant and resuspend the cells with 20 ml of DMEM and count the cells.
  7. Resuspend 2 millions spleen cells in 50 μl of 1:200 Fc block in FACS buffer and incubate for 30 min on ice.
  8. Wash the cells with 200 μl of PBS and spin down the cells at 300 RCF for 5 min at 4 °C.
  9. Discard the supernatant and resuspend the cells with 30 μl of anti-mouse CXCR5-biotin (1:50 in FACS buffer) and incubate for 30 min on ice.
  10. Wash the cells with 200 μl of PBS and spin down the cells at 300 RCF for 5 min at 4 °C.
  11. Discard the supernatant and resuspend the cells with 30 ul of anti-Rat IgG-biotin (1:50 in FACS buffer) and incubate for 30 min on ice.
  12. Wash the cells with 200 μl of PBS and spin down the cells at 300 RCF for 5 min at 4 °C.
  13. Discard the supernatant and resuspend the cells with 30 μl of the mixture of antibody 3-7 (1:50 in FACS buffer) and incubate for 30 min on ice.
  14. Wash the cells with 200 μl of PBS and spin down the cells at 300 RCF for 5 min at 4 °C.
  15. Resuspend the cells in 400 μl DAPI (3 μM in PBS) and analyze the cells using BD LSR II flow cytometer.

Gating strategy

  1. Gate on lymphocyte gate



  2. Gate on CD4 positive population within the lymphocyte gate



  3. Exclude B220 and CD11b positive cells from CD4 positive gate



  4. Gate on Tfh population based on CXCR5 and PD-1 expression


Recipes

  1. FACS buffer
    3% fetal bovine serum in PBS

Acknowledgments

This protocol was developed or modified in Dr. Anne Davidson’s lab at Feinstein Institute for Medical Research, NY, USA. This work was supported by grants from the NY SLE Foundation (RB), Rheuminations, NIH AI082037 and AR 049938-01, NIH (PO1 AI51392 and the Flow Cytometry and Protein Expression and Tetramer Cores of PO1 AI51392).

References

  1. Liu, Z., Bethunaickan, R., Huang, W., Lodhi, U., Solano, I., Madaio, M. P. and Davidson, A. (2011). Interferon-alpha accelerates murine systemic lupus erythematosus in a T cell-dependent manner. Arthritis Rheum 63(1): 219-229.
  2. Ramanujam, M., Wang, X., Huang, W., Liu, Z., Schiffer, L., Tao, H., Frank, D., Rice, J., Diamond, B., Yu, K. O., Porcelli, S. and Davidson, A. (2006). Similarities and differences between selective and nonselective BAFF blockade in murine SLE. J Clin Invest 116(3): 724-734.

简介

生发中心是B细胞经历体细胞超突变和类别转换的主要位点。 最近发现的称为T滤泡辅助(TH)细胞的T细胞子集是生发中心反应所必需的,因此近年来已经成为深入研究的主题。 这个协议描述了一种可靠的方法来染色这个群体进行流动分析。 这个协议是在费恩斯坦医学研究所的Anne Davidson博士的实验室中开发或修改的。

关键字:流式细胞仪, 小鼠, 滤泡辅助性T细胞, 生发中心

材料和试剂

  1. 抗体
    1. 大鼠抗小鼠CXCR5-生物素(BD Biosciences,Pharmingen TM,目录号:551960)
    2. 山羊抗大鼠IgG-生物素(BD Biosciences,Pharmingen TM,目录号:554014)
    3. 链霉亲和素-PE Cy7(BD Biosciences,Pharmingen TM,目录号:557598)
    4. 大鼠抗小鼠B220-太平洋蓝450(eBioscience,目录号:48-0452-82)
    5. 大鼠抗小鼠CD11b-太平洋蓝450(eBioscience,目录号:48-0112-82)
    6. 大鼠抗小鼠CD4-APC eFluor 780(eBioscience,目录号:47-0042-82)
    7. 仓鼠抗小鼠PD-1-PE(eBioscience,目录号:12-9985-82)
    8. 小鼠Fc嵌段(BD Biosciences,Pharmingen TM,目录号:553141)
  2. 其他材料
    1. 源自鼠脾样品的单细胞悬浮液
    2. 胎牛血清(FBS)(Hyclone)
    3. Dulbecco改良Eagle培养基(DMEM)(Life Technologies,Invitrogen TM)
    4. 氯化铵(0.17M,过滤,高压灭菌)(pH7.4)
    5. DAPI核酸染色(Life Technologies,Invitrogen TM)
    6. FACS缓冲区(参见配方)

设备

  1. BD LSR II流式细胞仪
  2. 圆锥管(Corning)

程序

  1. 收获脾脏,通过用5ml的DMEM中的一对显微镜载玻片的磨砂表面轻轻地捣碎脾脏片段来产生单细胞悬浮液。
  2. 转移细胞到50毫升锥形管,并在300 RCF下旋转5分钟在4℃的细胞。
  3. 弃去上清液,不要打扰沉淀。
  4. 用5ml 0.17M氯化铵重悬细胞,并将细胞在冰上保持5分钟
  5. 加入15毫升DMEM的细胞,并在300 RCF在4℃下旋转5分钟
  6. 弃去上清液并用20ml DMEM重悬细胞并计数细胞
  7. 在FACS缓冲液中,在50μl的1:200Fc块中重悬2百万个脾细胞,并在冰上孵育30分钟。
  8. 用200μlPBS洗涤细胞,并在300 RCF下,在4℃下旋转5分钟
  9. 弃去上清液,并用30μl抗小鼠CXCR5-生物素(在FACS缓冲液中1:50)重悬细胞,并在冰上孵育30分钟。
  10. 用200μlPBS洗涤细胞,并在300 RCF下,在4℃下旋转细胞5分钟
  11. 弃去上清液,并用30ul抗大鼠IgG-生物素(在FACS缓冲液中1:50)重悬细胞,并在冰上孵育30分钟。
  12. 用200μlPBS洗涤细胞,并在300 RCF下,在4℃下旋转细胞5分钟
  13. 弃去上清液并用30μl抗体3-7的混合物(在FACS缓冲液中1:50)重悬细胞并在冰上孵育30分钟。
  14. 用200μlPBS洗涤细胞,并在300 RCF下,在4℃下旋转细胞5分钟
  15. 重悬细胞在400微升DAPI(3微米在PBS中),并使用BD LSR II流式细胞仪分析细胞。

门控策略

  1. 在淋巴细胞门上的门



  2. 门对淋巴细胞门内的CD4阳性群体



  3. 从CD4阳性门中排除B220和CD11b阳性细胞



  4. 基于CXCR5和PD-1表达的Tfh群体的门控


食谱

  1. FACS缓冲区
    3%胎牛血清的PBS溶液中

致谢

该方案在Anne Davidson博士在Feinstein Institute for Medical Research,NY,USA的实验室中开发或修改。 这项工作得到NY SLE基金会(RB),Rheuminations,NIH AI082037和AR 049938-01,NIH(PO1 AI51392和PO1 AI51392的流式细胞术和蛋白质表达和四聚体核)的赠予的支持。

参考文献

  1. Liu,Z.,Bethunaickan,R.,Huang,W.,Lodhi,U.,Solano,I.,Madaio,M.P.and Davidson,A。(2011)。 干扰素-α以T细胞依赖的方式加速小鼠系统性红斑狼疮。 arthritis Rheum 63(1):219-229。
  2. Ramanujam,M.,Wang,X.,Huang,W.,Liu,Z.,Schiffer,L.,Tao,H.,Frank,D.,Rice,J.,Diamond,B.,Yu,KO,Porcelli ,S.and Davidson,A。(2006)。 鼠科SLE中选择性和非选择性BAFF阻断之间的相似性和差异。 Clin Invest 116(3):724-734
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引用:Liu, Z. (2012). FACS Staining for Follicular Helper T Cells. Bio-protocol 2(2): e35. DOI: 10.21769/BioProtoc.35.
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Jawaher Alsughayyir
Univ of Cambridge
Thanks for sharing the protocol, I noticed that in the washing (steps 8, 10, 12, 14) you used 200 mL of PBS. Is this correct? If so, what tubes did you use for containing such amount of washing solution. Thx
12/4/2014 3:21:29 PM Reply
Zheng Liu
The Feinstein Institute for Medical Research, Manhasset, USA

It is a typo. It was meant to be 200ul. Thank you for your question and we apologize for the error.

12/5/2014 5:07:31 AM


put the info on the additon of ALL the antibodies, and make a comment on the exlusion of different fluorochromes, Alex
8/20/2012 1:16:57 PM Reply
Zheng Liu
The Feinstein Institute for Medical Research, Manhasset, USA

Dear Alex:

Thank you for your question. Please clarify WHAT info you want regarding the addition of WHICH antibodies as the steps to add antibodies into the samples were described in the protocol with great details. Also I am not exactly sure what you mean by "exclusion of different fluorochromes". Were you asking what fluorochromes cannot be used? Or the compensation of them?

Please elaborate on your question, thank you.

8/20/2012 6:52:29 PM