Easysep rhesus cd4 t cell isolation kit

After 7 days of incubation, co-cultured cT_FH cells and cognate circulating B cells, both obtained from necropsy samples of six chronically infected macaques (all recipients of mock-vaccine) and six protected animals (three vaccinated and three recipients of mock vaccine) were harvested and cT_FH cells were magnetically sorted using the EasySep Rhesus CD4⁺ T cell isolation kit (STEMCELL, Cambridge, MA). Magnetically sorted cT_FH cells were treated with BD GolgiPlug and BD GolgiStop for 1 h at 37°C in the presence of 5% CO₂, and stained with Live/Dead aqua dye followed by surface staining antibodies for cT_FH phenotyping (Table 1) and permeabilized with the Cytofix/Cytoperm permeabilization kit (Becton-Dickson, Cockeysville, MD, USA). Intracellular staining of IL-21 and BAFF was carried out using the ICS antibodies listed in Table 1. Cells were washed and fixed with 2% PFA and saved at 4°C until acquisition in on an18-parameter BD LSRII (BD Biosciences) and analyzed with FlowJo 10.2.

Viably frozen PBMCs obtained from three macaques prior to immunization, 14 infected macaques 40 weeks post-infection, and nine protected macaques 2 weeks following the last challenge were thawed and used for either negative selection of CD4⁺ T cells (~ 20–40 million cells) or positive selection of B cells (~ 10–20 million cells). CD4⁺ T cells were negatively sorted using the EasySep Rhesus CD4⁺ T cell isolation kit (STEMCELL, Cambridge, MA), stained with anti-CXCR5-PE (Nonhuman Primate Reagent Resource), treated with anti-PE microbeads (Miltenyi Biotech) and positively isolated using a magnetic column. For B cell positive selection, cells were labeled with anti-CD19 magnetic beads (Miltenyi Biotec) and isolated using a magnetic column. To check the purity of the enriched cT_FH cells and B cells obtained by this procedure, PBMC obtained from three additional necropsied macaques were stained with Alexa700 anti-CD3 (SP34-2), BV711 anti-CD4 (L200), FITC anti-CD8 (RPA-T8) (BD Bioscience), BV650 anti-CD20 (2H7) (BioLegend), BV605 anti-CD16 (CB16) (eBioscience), and PE-Cy5 anti-CD19 (J3-119) from Beckman Coulter. The cells were acquired on a BD LSRII (BD Biosciences) and analyzed as described above.