Pe anti rat cd8a

After 31 days, the spleens were harvested and the animals were killed with an overdose of sodium thiopental. Their spleens were weighed and single suspensions were prepared from them by gently squashing them with a syringe plunger and 10 ml of phosphate-buffered saline (PBS) solution in a Petri dish. The mixture obtained was filtered to obtain single-cell suspensions that were then centrifuged at 300×g to collect cell pellets. After the pellets had been fixed, they were resuspended in PBS with 2% benzenesulfonic acid. We made two samples of resuspended cells from each rat’s spleen and separately incubated each for 30 min with an excess of fluorescein isothiocyanate-conjugated mouse anti-rat CD4 monoclonal antibody and PE-anti-rat CD8a (both from BioLegend, San Diego, CA, USA). We identified lymphocyte subsets using a flow cytometer (FACScan; Becton Dickinson, Mountain View, CA, USA). The positive cells were expressed as a percentage of the 10,000 cells counted.

After stressed 1d, 3d, 5d and 7d, 100 μl samples of anti-coagulated whole blood were collected. The whole blood was then stained with FITC anti-rat CD4 (#201505, Biolegend, USA) and PE anti-rat CD8a (#200608, Biolegend, USA) for 15–20 min, while shielded from light at room temperature of 22 ± 2°C. After incubation, the samples were fixed with 2ml 1X red blood cell (RBC) lysis buffer (#420301, Biolegend, USA) for 10 min, while shielded from light at room temperature, before being centrifuged at 350g for 5 min. The resulting supernatant was discarded, and cells were washed and resuspended in at least 2 ml of cell-staining buffer for flow cytometry analysis (Beckman CytoFLEX, USA).

To detect T cell subsets of CD4⁺ and CD8⁺, the cells were adjusted to a density of 1 × 10⁷ cells/ml. Then, 100 ul cells from each sample were centrifuged at 1500 rpm for 5 min and suspended with PBS. After centrifugation at 2000 rpm for 2 min at 4 °C, the cells were washed twice with PBS, containing 0.5% bovine serum albumin (BSA). The cells were stained at 4 °C for 30 min with APC-anti-rat CD3 (Invitrogen), FITC-anti-rat-CD4, and PE-anti-rat-CD8A (Biolegend, San Diego, CA, USA). The stained cells were suspended in 0.5 ml of PBS containing 0.5% BSA and analyzed by flow cytometry.