Hamster cd3e apc

To obtain the suspension of mononuclear cells and granulocytes, whole blood samples were centrifuged at 400× g for 30 min at RT using a gradient density Ficoll Paque Premium (GE Healthcare, Chicago, IL, USA) and the samples were subsequently frozen. The single-cell suspension of spleen and lymph nodes was prepared by passing through sterile nylon filters on a petri dish and then centrifuged twice at 192× g for 7 min at 4 °C. The cells (1 × 10⁶) suspended in phosphate buffered saline (PBS) with 2% fetal bovine serum (FBS; GE Healthcare, Chicago, IL, USA) were incubated with CD16/CD32 antibody to block the Fc receptors. After incubation, the splenocytes, mononuclear blood cells, and lymph nodes were stained with the anti-mouse conjugated antibodies as follows: rat CD8a-BV510, rat CD4-PE-Cy7, rat CD19-PE, hamster CD3e-APC, rat CD25-BV421, and rat CD335(NKp46)-FITC (BD Biosciences, Franklin Lakes, NJ, USA). Granulocytes were stained with rat CD45-PerCP-Cy5.5, rat Ly6G-Ly6C-APC, rat CD184-PE, and hamster CD54-FITC (BD Biosciences, Franklin Lakes, NJ, USA). Prior to analysis, the cells were washed with PBS containing 2% FBS (centrifuged at 192× g for 7 min at 4 °C). For data analysis, a BD LSR Fortessa cytometer with FACSDiva V8.0.1 software (BD Biosciences, Franklin Lakes, NJ, USA) was used.

The single-cell suspension of spleen cells from young and aged mice (1×10⁶) resuspended in PBS with 2% FBS (GE Healthcare, Chicago, IL, USA) was incubated with CD16/CD32 antibody to block the Fc receptors. After incubation, splenocytes were stained with the anti-mouse conjugated antibodies as follows: rat CD8a-BV510, rat CD4-PE-Cy7, rat CD19-PE, hamster CD3e-APC, rat CD25-BV421, and rat CD335(NKp46)-FITC (BD Biosciences, Franklin Lakes, NJ, USA). Prior to the analysis, the cells were washed with PBS containing 2% FBS (192×g at 4°C). For data analysis, a BD LSR Fortessa cytometer with FACSDiva V8.0.1 software (BD Biosciences, Franklin Lakes, NJ, USA) was used.