Fluorescently labeled monoclonal antibodies

Splenocytes were obtained by gentle disruption of the spleen through sieve mesh. After the lysis of spleen tissues with 1× RBC Lysis Buffer (Roche, Basel, Switzerland), remaining cells were re-suspended in RPMI-1640 medium with 10% fetal bovine serum, 100 µg/mL streptomycin and 100 IU/mL penicillin, followed by centrifugation and washing with phosphate buffered saline (WELGENE, Gyeongsan, Korea).
FACS was used to analyze sub types of the isolated splenocytes. Briefly, spleen cells were harvested and washed with 1× Dulbecco’s phosphate buffered saline (DPBS, WELGENE, Gyeongsan, Korea). The cells were blocked with 1 µg anti-mouse IgG solution in PBS for 15 min at 4 °C, and then stained with fluorescently labeled monoclonal antibodies (Biolegends, San Diego, CA, USA) for an additional 15 min at 4 °C. Monoclonal anti-bodies were directly labeled with the following fluorescent tags; CFSE for Live/Dead cell, PE-CD3 for total T cell, APC-CD4 for helper T cell, Pacific-Blue-CD8a for cytotoxic T cell and regulatory T cell, and PE/Cy7-CD45R/B220 for B cell. After centrifugation, DPBS was added to the cells, and twenty thousand viable cells per treatment (as determined by light scatter profiles) were analyzed using a BD FACS LSR Fortessa flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).