Live death vivid detection kit

Fresh PBMCs were isolated by density gradient centrifugation. FACS staining was carried out in PBS 1X supplemented with 2% FBS (foetal bovine serum) for 30 min at 4°C using the following fluorochrome-conjugated antibodies: APC-Vio770 anti-CD4, PerCP-Cy5.5 anti-CD3 (Milteniy Biotech, Paris, France), PE-CF594 anti-CD45RO, Alexa-Fluor 647 anti-CCR4, PE anti-CXCR3, PE-Cy7 anti-CCR6 (Becton Dickinson Biosciences, Pont de Claix, France). Intracellular staining was carried out using the FoxP3 permeabilization solution kit according to the manufacturer’s instructions (eBioscience, Paris, France) together with Alexa-Fluor 488 anti-SAMHD1 (from O. Schwartz’s lab; Institut Pasteur, Paris) and PE anti-Ki67 (Becton Dickinson). Dead cells were excluded using the Live/Death Vivid detection kit labeled with an aqua dye (Invitrogen). Fluorescence intensities were measured with an LSR II flow cytometer (Becton Dickinson) and analyzed using FlowJo version 10.0.7 (Tree Star Inc., Ashland, Oregon, USA).

Multi-color flow cytometry was performed on PBMC by acquiring 106 formaldehyde-fixed cells at the Gallios (Beckman Coulter) flow cytometer, and data analysed with FlowJo 8.8.7 (Treestar Inc.). The following mouse antihuman fluorochrome-conjugated monoclonal antibodies were used: CD19-PER-CP-Cy5.5 (SJ25-C1), CD10-PE-Cy7 (Hl10a), CD38-APC (HIT2), CD21-PE (B-ly4), CD27-V450 (M-T271), IgD-APC-H7 (IA6-2) and IgM-FITC (G20-127) (BD Biosciences). Dead cells were excluded using the Live/Death Vivid detection kit labeled with a near-infrared dye (Invitrogen, Carlsbad, California, USA). Gating strategy was performed as follows: only singlets were acquired and live B cells were gated on Vivid-CD19+ lymphocytes. Only samples with viability above 85% were subjected to the B-cell analysis. Transitional (“TR”) B cells were identified as CD19+CD27-CD10+. Thereafter, the CD10- cell population was gated on CD27++CD38high+ to identify Plasma-cells. Whereas on the CD27+CD38high- gate the following populations were identified: naive (“NV” CD27-CD21high+), resting memory (“RM” CD27+CD21high+), activated memory (“AM” CD27+CD21-) and tissue-like memory (“TLM” CD27-CD21-). RM, AM and TLM were each further characterized on the basis of Ig-expression into switched (“SW”, IgM-IgD-), Marginal Zone-like (“MZL”, IgM+IgD+), IgM-expressing (“IgM+”, IgM+IgD-) and IgD-expressing (“IgD+”, IgM-IgD+).