Pd 1 bv785

Cryopreserved spleen cells from uninfected and HIV infected hDRAGA mice were thawed and cultured overnight in R-15 media (RPMI 1640 supplemented with 15% heat inactivated FBS (Sigma), 1X nonessential amino acids (Gibco), 1X Glutamax (Gibco), 1X Primocin (In vivogen), and 100 units Benzonase (Sigma)). Cells were centrifuged at 300xg for 10 minutes, then resuspended in an antibody cocktail consisting of CD3-AF700 (clone SP34-2; Becton Dickinson (BD)), CD19-PeCy7 (Clone SJ25C1; Tonbo), IgD-FITC (Clone IA6-2; BioLegend), CD8-BV510 (clone RPA-T8; BD); CXCR5-PE (clone MU5UBEE; Invitrogen), PD-1-BV785 (clone EH12.2H7; BioLegend), CD38-APC (clone HIT2; Tonbo), and Brilliant Stain Buffer (BD). Cells were labeled for 20 minutes at room temperature in the dark, then washed with PBS without calcium and magnesium, then centrifuged at 300xg for 10 minutes. Pellets were resuspended in PBS supplemented with 2% FBS and 1mM EDTA and 7-AAD (Tonbo) was added. Cells were passed through a 40 μm filter cap and analyzed on an LSR Fortessa (BD) flow cytometer.

The following fluorochrome conjugated anti-human antibodies were used: CD3 APC efluor 780 (eBioscience #47-0036-42) or BV650 (Biolegend #317324), CD4 BV 510 (BD Horizon/BD Biosciences #562970), CD8 evolve 655 (eBioscience #86-0088-42), CD33 APC (eBioscience#17-0338-42), CD56 efluor 710 (eBioscience #46-056-42) or FITC (Biolegend #304604), PD-1 BV 785 (BioLegend #329930), CCR7 BV785 (Biolegend #353230), CD19 BV785 (Biolegend #302240), CD62L BV785 (Biolegend #304830), IFN-γ efluor 450 (eBioscience #48-7319-42), Perforin Alexa Fluor 647 (Biolegend #353322) or PE (Biolegend #353304), granzyme FITC (Biolegend #515403). Appropriate isotype control antibodies were employed to determine the specificity of test antibodies.