The extirpated spleen was removed in germ-free condition, and gently grinded in aseptic phosphate-buffered saline (PBS) through a stainless steel meshes. The splenocyte suspensions were resuspended in erythrocyte lysis buffer for 5 min to remove the red blood cells. After centrifuged at 300 g for 5 min, cell numbers and viability (over 95%) were assessed microscopically using trypan blue dye exclusion technique.