Cd5 percp cy5

The CLL cells from all the blood samples were stained with optimal concentrations of antibody combinations and directed against the following surface antigens: CD183(CXCR3)-FITC, CD20-PE, CD5-PerCP-Cy5.5, CD38-Pe-Cy7, CD49d-APC, CD19-APC-Cy7, CD184 (CXCR4)-BV421 and HLA-DR-BV510 (all procured from BioLegend), as previously reported [5 (link),15 (link)]. Isotype-matched antibodies (BioLegend) were used as negative controls.
The determination of s-CLL and l-CLL cells was conducted using FSC data and a back-gating strategy. The analysis was performed using a BD FACSCanto II (Becton Dickinson) instrument, and data acquisition was performed using BD FACSDiva software (v.8.0.2; Becton Dickinson). Flow cytometry data were analysed using FlowJo v.X0.7 software (Tree Star, Inc., San Carlos, CA, USA). In all the experiments, a minimum of 10,000 events was counted. The results were expressed as a percentage and mean fluorescence intensity (MFI).

Blood was collected in EDTA tubes, and 100 μl was incubated with anti-human CD19-eFluor450 (eBioscience, San Diego, CA, USA), CD24-fluorescein isothiocyanate (FITC) (BD biosciences, Franklin Lakes, NJ, USA), CD27-APC-eFluor780 (eBioscience), CD38-PeCy5 (eBioscience), CD5-PerCp-Cy5.5 (Biolegend, San Diego, CA, USA) or the corresponding isotype controls. After 15 minutes cells were treated with fluorescence-activated cell sorting (FACS) Lysing solution (BD Biosciences). Samples were measured using an LSR-II flow cytometer (BD biosciences) and data were analyzed using Kaluza 1.2 flow analysis software (Beckman Coulter, Brea, CA, USA). B cells were divided into transitional, memory, naive, CD24^highCD38^high and CD24^highCD27+ B cells as described previously [14 (link)]. CD5+ B cells were gated on an isotype control.