Cd8 rpe

The splenic lymphocytes were collected for the measurement of the CD4+/CD8+ ratio as previously described^{27 (link)}. Briefly, 1 × 10⁶ splenic lymphocytes were incubated with anti-chicken CD3-SPRD, CD4-FITC and CD8-RPE (Southern Biotech, USA) at 4 °C for 30 min. Subsequently, lymphocytes were washed 3 times with phosphate buffer saline containing 1% fetal bovine serum, then resuspended and analyzed by FacsCalibur and CellQuest software (Becton Dickinson, USA). Viable lymphocytes were calculated based on light scatter (forward and side scatter) characteristics, and 10,000 events were analyzed for positive staining with SPRD, FITC and RPE antibodies.

Post-booster immunization, variations in the JOL1587-specific helper (CD3 + CD4+) and cytotoxic (CD3 + CD8+) T cell populations were analyzed following ex vivo stimulation of peripheral blood mononuclear cells (PBMCs) with soluble antigen extracted from wild-type S. Senftenberg. PBMCs were separated from blood samples of five chickens from each group (A, B and C) at 1 week post-prime and booster immunization. The isolated PBMCs were stimulated ex vivo with soluble antigen extracted from wild-type S. Senftenberg. The stimulated PBMCs were stained with anti-CD3, anti-CD4 and anti-CD8 antibodies, as described elsewhere [15 ]. Briefly, a total of 1 × 10⁶ PBMCs were stained with appropriately diluted mouse anti-chicken CD3-FITC, CD4-APC and CD8-RPE (Southern Biotech, USA). After incubation, all stained samples were washed three times with PBS and analyzed with a flow cytometer (Miltenyi Biotech, Germany). A total of 10,000 events were recorded and analyzed using the FlowJo single cell analysis software.