Clinical Research Directory

4-Evaluation of the Frequency of regulatory B cells (Bregs) by Flow Cytometry Using flow cytometry, circulating Bregs will be detected using FITC-conjugated-CD38, PE-conjugated-CD24 (Bioscience, USA), and PerCP-conjugated CD19 (BD Bioscience, USA). Briefly, 100 µl of blood sample will be incubated with 10 µL of CD24, CD38 and CD19 for 20 minutes at 4 °C in the dark. Following incubation, RBCs will be lysed and washed. Cells will be fixed and permeabilized then stained with APC-conjugated IL-10 (BD Bioscience, San Jose, CA, USA) and analysis will be done by FACS Calibur flow cytometry with CellQuest software (Becton Dickinson Biosciences, San Jose, CA, USA). An isotype-matched negative control will be used for each sample. Forward and side scatter histograms will be used to define the lymphocytes population. CD19+ IL-10+ B cells will be gated, then the expression of CD38 and CD24 on the CD19+B cells will be detected. Bregs will be identified as CD19+ IL-10+CD24+hiCD38+hi cells.