Cd38 percpcy5.5 clone hit2

PBMC from healthy donor volunteers isolated from fresh buffy coats were incubated with 1μM IPP and 100U/mL IL-2 or 100U/mL IL-2 alone. Expression of CD4 and CCR5 receptors along with expression of activation markers HLA-DR, CD25 and CD38, was controlled by flow cytometry at days 0 and six of culture. mAb used were; CD4-FITC (clone RPTA-4, BD), Vδ2-PE (clone B6, Biolegend), CCR5-V450 (clone 2D7/CCR5, BD), HLA-DR-FITC (clone TU36, BD), CD25-V450 (clone M-A251, BD) and CD38-PerCPCy5.5 (clone HIT2, BD). Briefly, cells were blocked with FBS (Sigma) for 10 minutes on ice, resuspended in staining buffer (PBS-2% FBS), incubated for 20 minutes on ice in the dark with combinations of the mAb, or suitable isotype controls, and washed twice. Cells were then fixed with 2% paraformaldehyde solution and analyzed in the Attune acoustic cytometer (Applied Biosystems).

Single-cell suspensions from the BM and peripheral blood were first incubated with human BD Fc block (BD Biosciences) to block Fcγ receptor and then stained with the following anti-human Abs: CD19-APC (clone HIB19, BioLegend), CD34-PE (clone 563, BD Biosciences), CD20-FITC (clone 2H7, BioLegend), CD27-APC/Cyanine7 (clone O323, BioLegend), IgD-PE (clone IA6-2, BD Biosciences), IgM-FITC (clone G20-127, BD Biosciences), CD43-PE (clone CD43-10G7, BioLegend), CD38-PE/Cy7 (clone HIT2, BioLegend), CD38-Percp/Cy5.5 (clone HIT2, BD Biosciences), CD24-PE (clone ML5, BD Biosciences), CD27-V450 (clone M-T271, BD Biosciences), IgD-BV510 (clone IA6-2, BD Biosciences), IgG-PE/Cy7 (clone G18-145, BD Biosciences), Ki-67–PE (clone Ki-67, BioLegend), CD80-FITC (clone L307.4, BD Biosciences), CD86-PE (clone IT2.2, BD Biosciences), HLADR-PE (clone G46-6, BD Biosciences) and CD69-PE/Cy7 (clone FN50, BD Biosciences). 7-AAD Viability Staining Solution (eBioscience, Thermo Fisher Scientific) was used for live versus dead cell discrimination. Samples were analyzed with a FACSVerse flow cytometer (BD Biosciences) using the FACSuite software. Data analysis was performed with FlowJo 10 software (Treestar).