Cd27 pecy7

Cells from heparinized blood were phenotypically analyzed in a 10-color MoAb conjugate combination using a Navios™ instrument with 10-color PMTs and three solid-state lasers (Beckman Coulter, Fullerton, FL). The list mode data files were further analyzed using Kaluza™ software (Beckman Coulter). In order to guarantee that the optics, laser, fluidics and fluorescence intensity were stable during all measurements calibration was performed using Flow Check Pro Fluorospheres (Beckman Coulter) and Cyto-Cal Multifluor + Violet Fluorescence Alignment Beads (Thermo Scientific, Fremont, CA, USA). After erythrocyte lysis (BD Pharm-Lyse, BectonDickinson) cells were washed with PBS with 1 % bovine serum albumin before being labeled with fluorochrome-conjugated mAbs. After incubation for 30 minutes at 4 °C in the dark, cells were washed twice to remove unbound antibodies and analyzed. For cell surface staining, the following mAbs were used: IgD-FITC, IgM-PE (both Dako, Denmark) and CD3-ECD, CD4-PECy5.5, CD27-PECy7, CD20-PacB, CD45-KromeOrange, CD56-APC, CD8-APC-Alexa Fluor700 and CD19-APC-Alexa Fluor750 (all Beckman Coulter, Marseille, France). Subsequently, the various lymphocyte subpopulations were analyzed on the flow cytometer using CD45/SSC to gate the lymphocyte population.

Phenotypic analysis of cell suspensions was performed by flow cytometry. Cells were stained with a panel of seven 8-colour antibody combinations (Table 1). The following 6 monoclonal antibodies were used as a “backbone” in all combinations: CD38-PerCP5.5, CD10-APC, CD19-APC-H7, CD5-V450, CD45-V500 (BD Biosciences) and CD27-PeCy7 (Beckman Coulter). All FITC- or PE-labelled antibodies were specific of a particular combination, i.e. CD20-FITC, CD44-FITC, CD24-PE, CD40-PE, (Beckman Coulter); CD43-FITC, CD81-FITC, CD86-FITC, CD21-PE, CD22-PE, CD23-PE, CD268 (BAFF-R)-PE, IgD-PE, (BD Biosciences) IgM-FITC (Dako). Clone and isotype specificity of these antibodies are detailed in S1 Table; the antibodies were used at the dilution recommended by the manufacturers.
Samples containing 10⁶ cells in a volume of 100 μl were incubated with the combination of antibodies at the supplier recommended concentration during 20 minutes in the dark, at 4°C. After erythrocytes lysis with NH₄Cl at 4°C for 5 minutes, samples were washed with HBSS medium (Gibco).
Analysis was performed using 3-laser, 8-colour BD FACSCanto II flow cytometer (BD Biosciences) and FACSDiva software version 6 (BD Biosciences). At least 10⁴ cells were acquired in the CD19⁺ gate. BD CompBeads (BD Biosciences) were used for compensation settings. Cytometer performances were checked daily using CST beads (BD Biosciences).