Cd23 pe cy7

PBMCs were isolated from whole blood using Ficoll (Lymphocyte^® Cell Separation Media, Mediatech) gradient centrifugation. Cryopreserved PBMC from HIV-infected subjects were used in the immunophenotyping experiments. The cells were stained with FcRL4-APC and CD19-PE-Texas Red and CD19⁺FcRL4^pos and CD19⁺FcRL4^neg B cells were FACS purified and cultured overnight in the presence of 10 µg/ml CpG-B ODN2006 (TLR9L), 2 µg/ml PAM3CSK4 (TLR2L), or 2 µg/ml Imiquimod (InvivoGen). B cells (CD19⁺) from healthy controls were purified from PBMC using the B Cell Isolation Kit II (Miltenyi Biotec) and AUTOmacs (Miltenyi Biotec). After 4H, the cultures of CD19⁺ B cells were supplemented with Brefeldin A (1:1,000, BD). After overnight incubation, the cells were surface stained (CD23-PE-Cy7, BD Biosciences), fixed/permeabilized (Fix/Perm Kit BD Biosciences), and stained for intracellular IL-6 (IL-6-PE, BD Biosciences). All samples were acquired on an LRSII (BD Biosciences) flow cytometer and the data analyzed using FlowJo software (Tree Star Inc.). Florescence parameters were normalized using Rainbow Calibration Particles (Spherotech) and antibody bound CompBead (BD Biosciences). Gating was determined by unstained controls.