Lymph nodes (cervical, axillary, branchial, and inguinal) and spleens were harvested from the dissected mice. To obtain a cell suspension, they were then mashed through a 70-μm strainer with 1 ml syringe plunger into 5 ml cRPMI RPMI medium, (Cat# SH30027.01; HyClone) supplemented with 10% (vol/vol) FCS (Cat #SV30160.03; HyClone), 100 U/ml penicillin, 10 mg/ml streptomycin (Cat #SV30010; HyClone), 292 mg/ml L-glutamine (Cat #SH30034.01; HyClone), 1 mM sodium pyruvate (Cat #11360-070; Gibco), 1X MEM nonessential amino acids (Cat #11140-050; Gibco), 25 mM Hepes, pH 7.3 (Cat #SH30237.01; HyClone), and 50 μM 2-mercaptoehthanol (Cat #M3180; Sigma-Aldrich).
The splenocytes were subjected to red blood cell lysis using ammonium chloride-potassium lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) for 4 min at room temperature. Thereafter, the lysis was quenched by adding 5 ml cRPMI and pelleting down the cells at 300g, 5 min. Pellet was then resuspended using 5 ml cRPMI. Cells were counted using a Beckman Coulter Z1 particle counter.
The splenocytes were subjected to red blood cell lysis using ammonium chloride-potassium lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) for 4 min at room temperature. Thereafter, the lysis was quenched by adding 5 ml cRPMI and pelleting down the cells at 300g, 5 min. Pellet was then resuspended using 5 ml cRPMI. Cells were counted using a Beckman Coulter Z1 particle counter.