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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.

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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

免疫学 > 免疫细胞染色 > 流式细胞术
作者: Zheng Liu
Zheng LiuAffiliation: The Feinstein Institute for Medical Research, Manhasset, NY, USA
For correspondence: zl2119@caa.columbia.edu
Bio-protocol author page: a12
Vol 2, Iss 2, 1/20/2012, 10626 views, 2 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.35

[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(鼠标), 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.


How to cite this protocol: Liu, Z. (2012). FACS Staining for Follicular Helper T Cells. Bio-protocol 2(2): e35. DOI: 10.21769/BioProtoc.35; Full Text



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12/4/2014 3:21:29 PM  

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

12/5/2014 5:07:31 AM  

Zheng Liu (Author)
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.

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8/20/2012 1:16:57 PM  

put the info on the additon of ALL the antibodies, and make a comment on the exlusion of different fluorochromes, Alex

8/20/2012 6:52:29 PM  

Zheng Liu (Author)
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.

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