Cd39 bv421

The frequency of plasmablasts (PBs) was measured in the blood of 12 macaques vaccinated with ALVAC-SHIV/gp120 Alum before vaccination and 7 days after the last immunization. Cells were stained with CD3 (Clone SP34-2), CD14 (Clone M5E2), CD16 (Clone 3G8), and CD56 (Clone B159), all conjugated in ALEXAFluor700 (BD Biosciences; San Jose, California, USA), PE-Cy5-CD19 (Clone J3-119, Beckman Coulter; Brea, California, USA), QDOT-650-CD20 (Clone 2H7, eBioscience; San Diego, California, USA), FITC-CD38 (Clone AT-1, StemCell; Massachusetts, USA), BV421-CD39 (Clone MOCP-21, BioLegend; San Diego, California, USA), PE-Ki67 (Clone B56, BD Biosciences), and anti-CXCR3 (CXCR3/CD183 PE-CF594 conjugated). Clone 1C6BD (Biosciences cat. #11718; anti-α4β7) was kindly provided by Dr. Ansari through the NIH AIDS Reagent Program, Division of AIDS, NIAID. Cells were permeabilized with Cytofix/Cytoperm (BD Biosciences). Acquisition was performed on an LSRII cytometer (BD Biosciences) and data were analyzed by FlowJo software (TreeStar; Ashland, Oregon, USA). Plasmablasts were gated as previously described: lineage-(CD3^- / CD14^- / CD16^- / CD56⁺) / (CD19⁺/ CD20⁺ / (CD21^-CD27⁺) / Ki67^+/++ / (CD38^+/++ / CD39⁺) [18 (link)]. The frequency of PBs expressing CXCR3 or α₄β₇ was then calculated.

PBMCs were thawed and washed twice with PBS. Then, the cells were stained with the following anti-human antibodies: PE-Cy7-CD3, PE/Dazzle594-CD4, APC-Cy7-CD8a, PE-Cy7-CD14, PerCP-Cy5.5-CD16, BV421-CD39, PE-CD73 (BioLegend), anti-nitrotyrosine (anti-NT) rabbit (Cat. #BS-8551R, Bioss Thermo Fisher) and Zombie Aqua (BioLegend). Nitric oxide production was evaluated using the molecular probe DAF-FM DA (10 μM, Cat. #D23844 Invitrogen). The oxidized product was measured at excitation/emission wavelengths of 488/520 nm. Labeled samples were acquired using a BD LSRFortessa FACS cytometer, and data were analyzed using FlowJo™ v10 software (Tree Star, Inc.). The compensation matrixes were designed using UltraComp eBeads™ Compensation Beads (01-222-42; Invitrogen) with specific markers.

Cell surface and intracellular molecular expressions were evaluated by flow cytometry using CytoFLEX (BeckmanCoulter, U.S.A.). Fluorescein-conjugated mouse anti-human antibodies were used, including CD3-Alexa Fluor 700, CD3-BV650, CD8-BV786, CD8-PerCP/Cy5.5, CD45RA-APC/CY7, CCR7-PE/CY7,CD27-PE, CD28-BV421, CD69-APC/CY7, CD103-BV605, CXCR3-BV510, HLA-DR-APC, CD39-BV421, PD-1-PE, CD127-PE/CY7, CD62L-PE/CY7, Perforin-APC, Granzyme B-PE, IFN-γ-PE, and IL-4-APC (Biolegend, UK). For cell-surface staining, single-cell suspensions were stained on ice for 30 min in PBS with 1% fetal bovine serum (FBS). For intracellular staining, cells were fixed and permeabilized using the Fix/Perm kit (BD Biosciences, U.S.A.). To detect intracellular cytokines, CD8⁺T cells were stimulated for 6 h with phorbol 12-myristate 13-acetate (PMA; 1 μg/mL; Sigma) and ionomycin (1 μg/mL; Sigma), and 4 h with GolgiStop (1 μL/mL; BD Biosciences) in a round-bottom 96-well plate. Thereafter, cells were harvested, stained for surface expression, and then fixed and permeabilized for intracellular staining. Flow cytometry data was analyzed using FlowJo software (BD, UK) and CytoExpert software (Beckman Coulter, U.S.A.).