Isolation and Expansion of SIV-Reactive Memory B Cells

Cryopreserved PBMCs were thawed, washed, and stained with an antibody cocktail (1:100 dilution) of CD3 (clone SP34-2, BD Biosciences), CD4 (clone OKT4, Biolegend), CD8 (clone RPA-T8, BD Biosciences), CD14 (clone M5E2, BD Biosciences), CD20 (clone 2H7, Biolegend), IgM (MHM-88, Biolegend), IgG (clone G18-145, BD Biosciences) and fluorescently labeled biotinylated SIVmac239 SOSIP.664 Env at room temperature for 20 min in the dark. SIVmac239 Env trimer probes were labeled with two different fluorophores, from which SIVmac239 Env dual positive memory B cells (CD3⁻CD4⁻CD8⁻CD14⁻CD20⁺IgM⁻IgG⁺SIVmac239 SOSIP²⁺) were analyzed with BD FACSMelody or BD FACSFusion, and single-cell sorted, cultured, expanded in 384-well plates as described previously^{28 (link),29 (link)}. Briefly, sorted B cells were cultured with Iscove’s modified Dulbecco’s medium (IMDM) with GlutaMAX (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1× MycoZap Plus-PR (Lonza), 100 U/mL human IL-2 (Roche), 50 ng/mL human IL-21 (Invitrogen), 50 ng/mL human IL-4 (Miltenyi), 0.1 μg/mL anti-rhesus IgG (H + L) (BioRad), and irradiated 3T3msCD40L feeder cells. Flow cytometric data were subsequently analyzed using FlowJo (v10.7.1).