Quantification of Lymphocyte Subsets in PBMC

PBMC were isolated by density gradient centrifugation on Ficoll-Paque Plus (GE Healthcare, Uppsala, Sweden) as described previously [5] (link). Frequencies of lymphocyte subsets were estimated in PBMC by means of flow cytometry using mAb against CD19-PE.Cy7, CD4-APC, CD27-FITC, CD10-APC, IgD-PE, IgM-PE.Cy5, IgG-APC and CD138-APC (BD Pharmingen, San Jose, CA, USA). Non-specific fluorescence was established with APC-mouse IgG1κ, PE-Cy7-mouse IgG1κ, FITC-mouse IgG1κ, PE-mouse IgG2aκ, PE-mouse IgG1κ isotype controls. At least 200,000 events per sample were collected in a FACSAria II flow cytometer (BD Immunocytometry Systems) and analyzed using FlowJo software (Tree Star, Inc., Ashland, OR, USA). Lymphocytes were identified by forward and side-angle light scatter characteristics. At least 80,000 lymphocyte-gated cells were analyzed for each sample. Absolute numbers were estimated based on the frequencies of the cells in flow cytometry and lymphocytes in the differential leukocyte count (Sysmex K-800 Automated Hematology Analyzer, Sysmex America, Inc., Mundelein, IL, USA), and expressed as the number of cells per microliter of blood.