Cd45ro pe cy7 clone uchl1

PBMC were thawed in complete 10% FBS RPMI media (Millipore Sigma) with Benzonase® Nuclease (Millipore Sigma). CD4 T cells were isolated by magnetic bead negative selection with the EasySep CD4 isolation kit (STEMCell Technologies). Cells were stained for L/D-AQUA (Thermo Fisher) and then extracellular staining was done using: CD4-BV605 (clone RPA-T4, BD Bioscience), CD45RO-PE-Cy7 (clone UCHL1, BD Biosciences), CD8-APC (clone RPA-T8, BD), CXCR5-BV421(clone J252D4, Biolegend), and CD3-PE (SK7, Biolegend). Stained cells were sorted on a BD FACSAria™. 50,000 sorted CD45RO⁺CXCR5⁺ or CD45RO⁺CXCR5^- cells were then plated in a 96 U bottom plate. B cells from a non-related healthy control donor were isolated by magnetic bead negative selection with the EasySep B cell isolation kit (STEMCell Technologies). 50,000 B cells were added to the corresponding 96 U bottom plate in 10% FBS RPMI media (Millipore Sigma) with antiretroviral drugs were added to the culture (200nM raltegravir, 200nM lamivudine) (NIH AIDS reagent program). After 7 days of co-culturing in a 37°C incubator, B-cell differentiation was determined by flow cytometry and absolute cell numbers were quantified using counting beads (Thermo Fisher). Staining for B-cell differentiation was performed using antibodies listed in Supplementary Table 3, panel 5.

Peripheral blood samples (10–20 ml) from 20 healthy young adults (13 F/7 M, age range 22–35 years) were collected in heparinized tubes. Peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation of whole blood over a Ficoll-Paque gradient (Biochrom) and washed 4× with ice-cold RPMI1640 culture medium (Gibco). CD19+ B cells, CD14+ monocytes, CD3+ T cells, CD4+ CD25− Th cells, CD4+ CD25+ Th cells, CD8+ T cytotoxic cells, CD4+ CD45RA+ CD25− naive Th cells, and CD4CD45RO+ CD25− memory Th cells were isolated by cell sorting using a BD FACS Aria II flow cytometer (BD Biosciences). The sorting strategy is shown in Figure S1 Supplementary Material. The isolated cell populations were phenotyped and used when their purity reached >95%. The antibodies used for cell sorting and phenotyping were the mouse antihuman monoclonal antibodies (mAbs) CD3-APC-H7 (clone SK7), CD19-APC (clone HIB19), CD14-FITC (clone M5E2), CD4-APC (clone RPA-T4), CD8-FITC (clone HIT8a), CD25-PE (clone M-A251), CD45RA-APC-H7 (clone 5H9), and CD45RO-PE-Cy7 (clone UCHL1) (BD Biosciences), CD25-PC5 (clone B1.49.9) (Beckman Coulter). Fluorescence minus one controls were used to identify any background spread of fluorochromes and establish gating boundaries. The data were analyzed using the BD FACS DIVA software v.8.