40 μm sterile nylon mesh

A single-cell suspension of rat spleens was obtained by manual mincing and passing through a 40-μm sterile nylon mesh (Thermo Fisher Scientific, Pittsburgh, PA) with a 3-ml rubber syringe plunger. Cells were collected by centrifugation at 1500 rpm for 3 min. The supernatant was discarded, resuspended in PBS, and centrifuged. The supernatant was discarded, and the cells were resuspended in RBC lysis buffer (BioLegend, San Diego, CA) according to the manufacturer’s method. After removal of RBCs, splenocytes were washed twice with PBS, centrifuged, and resuspended in an appropriate volume of cell staining buffer (BioLegend), and viable cells were counted using the TC20 automated cell counter (Bio-Rad Laboratories, Hercules, CA) using the trypan blue staining method. Approximately 1 ml of rat whole blood was harvested by cardiac puncture using a 1-ml insulin syringe precoated with heparin and collected in tubes containing 100 U/ml heparin. RBC lysis was performed using 1× RBC lysis buffer (BioLegend) according to the manufacturer’s instructions. The cell pellet obtained in the final step was resuspended in staining buffer and viable cells were counted.