Our results show that a 5-day treatment of healthy individuals with G-CSF increases the count of circulating PCs by 6-fold, that of circulating B lymphocytes by 4-fold and that of circulating HSCs by 44-fold. Circulating PCs comprised both CD19+ CD20− CD38++ CD138− plasmablasts and CD19+ CD20−CD38++ CD138+ PCs. PB and leukapheresis AZD2014 research buy samples were
obtained from 26 healthy donors (age range 22–66 years) treated with G-CSF (10 μg/kg per day) for 5 days in order to collect HSCs for allograft. In concordance with French ethical law, cells that were not used for the patient’s treatment could be used for research with the donor’s written agreement. Leukapheresis was performed using a continuous flow blood cell separator (COBE Spectra version
4; CaridianBCT, Lakewood, CO). For each donor, a PB sample was obtained at the time at which the leukapheresis procedure was performed and both PB and leukapheresis samples were analysed. PB mononuclear cells (PBMCs) were obtained by density centrifugation using Lymphocyte Separation Medium (Lonza, Walkersville, MD) and analysed. PB from 11 healthy donors (in the absence of acute or chronic infection or recent vaccination) was purchased from the French Blood Centre (Toulouse, France). Abs conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), energy-coupled dye, peridinin chlorophyll protein (PerCP)-Cy5·5, PE-Cy7, Pacific Blue, allophycocyanin (APC) and APC-H7, specific for human CD19 (clone SJ25C1), CD27 (clone L128), CD29 [β1-integrin (ITGβ1), clone MAR4], CD38 (clone HIT2 or HB7), CD43 (clone 1G10), CD45 (clones 2D1 and DNA Methyltransferas inhibitor HI30), CD49d (ITGα4, clone 9F10), CD49e (ITGα5, clone SAM1), CD56 buy PLX4032 (N-CAM, clone B159), CD62L (clone DREG-56), CD70 (clone Ki-24), CD106 (VCAM-1, clone 51-10C9), CD117 (clone 104D2), CD184 (CXCR4, clone 12G5),
CCR2 (CD192, clone 48607), human leucocyte antigen (HLA)-DR, DP, DQ (clone Tu39), ITGβ7 (clone FIB504), anti-immunoglobulin light chain lambda (IgLCλ, clone JDC-12), anti-immunoglobulin light chain kappa (IgLCκ, clone TB 28-2), anti-immunoglobulin G (IgG) (clone G18-145), anti-IgM (clone G20-127), and KI-67 (clone B56) were purchased from Becton/Dickinson (BD) Biosciences (San Jose, CA); CD20 (clone B9E9), CD34 (clone 581), CD58 [lymphocyte function-associated antigen 3 (LFA-3), clone AICD58] and CD138 (clone B-A38) were obtained from Beckman Coulter (Fullerton, CA); CCR10 (clone 314305) was from R&D Systems (Minneapolis, MN), CD19 (clone HIB19) was from eBiosciences (San Diego, CA), and both anti-IgA (polyclonal goat antibody) and anti-IgG (polyclonal goat Ab) were from Southern Biotech (Birmingham, AL). Leukapheresis samples and PBMCs were labelled with Abs conjugated to various fluorochromes. The number of CD34+ cells was estimated by flow cytometry using the FC500 (Beckman Coulter) or FACSAria (BD Biosciences) flow cytometer. B lymphocytes and PCs were identified using a seven-colour combination of fluorochrome-conjugated Abs.