Anti cd8 5h10

After anesthesia, blood was collected via retro-orbital bleeding using sterile capillary tubes (Paul Marienfeld GmbH & Co. KG., Lauda-Königshofen, Germany). The blood samples (50 μL) were stained using fluorescence-activated cell sorting (FACS) antibodies at 4°C for 20 min; then, the red blood cells (RBCs) were removed using RBC lysis buffer (Sigma-Aldrich). The samples were washed once with FACS buffer (phosphate-buffered saline [PBS] with 1% bovine serum albumin [BSA] and 20 mM ethylenediaminetetraacetic acid) and fixed using 4% PFA. Additionally, lymphocytes (1×10⁶) from the spleen and thymus were incubated with FACS antibodies at 4°C for 20 min. The cells were rinsed once with FACS buffer and fixed using 4% PFA. Cells were then analyzed using flow cytometry (BD FACS Verse^TM; BD Biosciences, San Jose, CA, USA), and FlowJo software (BD Biosciences) was used for data analysis. FACS antibodies anti-CD3 (145-2C11), anti-CD4 (RM4-5, GK1.5), anti-CD8 (53-6.7), anti-CD44 (IM7), anti-CD25 (3C7), anti-CD45R/B220 (RA3-6B2), and anti-T cell receptor (TCR)-β (H57-597) were purchased from BD Biosciences. Anti-CD3 (17A2) antibody was purchased from BioLegend (San Diego, CA, USA). Anti-CD8 (5H10) and anti-CD62L (MEL-14) were purchased from Invitrogen (Carlsbad, CA, USA).