Anti mouse cd8 pe cy7

Mice were immunized as mentioned above. PBS-treated mice were used as the control. Spleens were excised from the mice 14 days after the first immunization. Splenocytes were separated and counted on a Guava easyCyte 8HT (Merck Millipore, Billerica, Massachusetts, USA) with Viacount reagent (Merck Millipore, Billerica, Massachusetts, USA). The densities of the splenocytes were adjusted to 2.0 × 10⁶ cells/ml in complete RPMI 1640 containing 10% FBS. Splenocytes from each mouse were added to 2 wells of a 24-well plate (4.0 × 10⁶ cells per well). For the tfRFP-stimulated groups, 50 μg/ml tfRFP was added to the medium. An equal volume of PBS was added to the control groups. The splenocytes were cultured at 37ºC for 3 days. IFN-γ secretion was inhibited by monensin (Biolegend, San Dieg, California, USA) for the last 4 h of the culture. Finally, collected cells were fixed and stained with anti-mouse CD3 APC/Cy7 (Biolegend, San Dieg, California, USA), anti-mouse CD4 APC (Biolegend, San Dieg, California, USA), anti-mouse CD8 PE/Cy7 (Biolegend, San Dieg, California, USA), and anti-mouse IFN-γ PE (eBioscience, San Diego, California, USA) for flow cytometric analysis. A rat anti-mouse IgG1 kappa PE antibody (eBioscience, San Diego, California, USA) was used as an isotype control.