Facsversa flow cytometer

For the evaluation of cell apoptosis, an annexin V-FITC apoptosis staining/detection kit (Abcam, UK) was used following the manufacturer's protocol. Data acquisition and analysis were performed using the FACSVersa flow cytometer (BD Biosciences, US).

For flow cytometry analysis of IL-10-producing CD19⁺ B_regs, cells in splenocytes were analyzed for the expression of surface markers using the following monoclonal antibodies: fluorescein isothiocyanate (FITC)-conjugated anti-CD19 (clone 1D3; BD Biosciences, San Jose, CA, USA) and phycoerythrin (PE)-conjugated anti-IL-10 (clone JES5-16E3; eBioscience, San Diego, CA, USA). For subsequent IL-10intracellular staining, the Cytofix/Cytoperm kit (BD Biosciences) was used according to the manufacturer's protocol (BD Biosciences). For the analysis of CD4⁺CD25⁺FoxP3⁺ T_reg cells, the mouse T_reg-staining kit was used according to the manufacturer's instructions (eBioscience). The stained cells were analyzed by 3-5 color flow cytometry using a FACSversa flow cytometer (BD Biosciences). The FlowJo software (Tree Star Inc., OR, USA) was used to analyze flow cytometry data. To determine background staining, non-reactive iso-type-matched control monoclonal antibodies (eBioscience) were used and gated to exclude ≥98% of non-reactive cells.