Materials and Reagents

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 )
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

BD LSR II flow cytometer
Conical tubes (Corning)

Procedure

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.
Transfer the cells into 50 ml conical tubes and spin down the cells at 300 RCF for 5 min at 4 °C.
Discard the supernatant with aspiration without disturbing the pellet.
Resuspend the cells with 5 ml of 0.17 M ammonium chloride and keep the cells on ice for 5 min.
Add 15 ml DMEM to the cells and spin at 300 RCF for 5 min at 4 °C.
Discard the supernatant and resuspend the cells with 20 ml of DMEM and count the cells.
Resuspend 2 millions spleen cells in 50 μl of 1:200 Fc block in FACS buffer and incubate for 30 min on ice.
Wash the cells with 200 μl of PBS and spin down the cells at 300 RCF for 5 min at 4 °C.
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.
Wash the cells with 200 μl of PBS and spin down the cells at 300 RCF for 5 min at 4 °C.
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.
Wash the cells with 200 μl of PBS and spin down the cells at 300 RCF for 5 min at 4 °C.
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.
Wash the cells with 200 μl of PBS and spin down the cells at 300 RCF for 5 min at 4 °C.
Resuspend the cells in 400 μl DAPI (3 μM in PBS) and analyze the cells using BD LSR II flow cytometer.

Gating strategy

Gate on lymphocyte gate
Gate on CD4 positive population within the lymphocyte gate
Exclude B220 and CD11b positive cells from CD4 positive gate
Gate on Tfh population based on CXCR5 and PD-1 expression

Recipes

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

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.
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.