Igg clone g18 145

Multiparameter flow cytometry was performed using a BD FACSFortessa cell sorter operated by BD FACSDiva software (BD Biosciences, San Jose, CA). PBMC from rhesus macaques or human donors were stained with directly conjugated monoclonal antibodies against human (rhesus macaque cross-reactive) CD3 (clone SP34-2), CD20 (clone L27), CD21 (clone B-ly4), CD27 (clone M-T7271), IgM (clone G20-127), and IgG (clone G18-145) (all purchased from BD Biosciences) or directly conjugated polyclonal goat antibodies against human (rhesus macaque cross-reactive) IgD and IgA (both purchased from Southern Biotech, Birmingham, AL). Staining for intracellular immunoglobulins was performed with the Fixation/Permeabilization solution kit (BD Biosciences) according to the manufacturer’s instructions after surface staining with the appropriate antibodies using protocols previously described [28 (link)]. Data were analyzed and illustrated using FlowJo software (Tree Star Inc., Ashland, OR). The FACS data generated from post SIV infection samples can be found at FlowRepository (ID: FR-FCM-ZZVV, FR-FCM-ZYZZ, and FR-FCM-ZYZY).

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).

Cryopreserved PBMCs were thawed, washed, and stained with an antibody cocktail 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 (clone G20-127, BD Biosciences or clone MHM-88, Biolegend), IgG (clone G18-145, BD Biosciences) and fluorescently labeled BG505 Env at room temperature for 20 min in the dark. BG505 SOSIP.664 was labeled with two fluorophores separately, from which dual positive Env-specific (CD3^-CD4^-CD8^-CD14^-CD20⁺IgM^-IgG⁺BG505 SOSIP²⁺) and/or 398F1 SOSIP⁺ memory B cells were analyzed with a FACSFusion sorter and then single-cell sorted, cultured, and expanded as previous described (Huang et al., 2013 (link)). In brief, cells were sorted into Iscove’s modified Dulbecco’s medium (IMDM) with GlutaMAX (GIBCO) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1X MycoZap Plus-PR (Lonza), 100 U/mL IL-2 (Roche), 50 ng/mL IL-21 (Invitrogen), 50 ng/mL IL-4 (Miltenyi), 0.1 μg/mL anti-rhesus IgG (H+L) (BioRad) and irradiated 3T3msCD40L feeder cells.