Flow Cytometric Analysis of Rat T Cells and DCs

Analysis of BTLA expression on CD4+ lymphocytes in peripheral blood mononuclear cells (PBMCs) of patients from the BPAR and stable groups was performed as previously described^{27 (link)}. Peripheral blood cells of rat recipients at D3 and D7 following kidney transplantation were collected and then stained with APC-labeled anti-CD3 (eBioscience CA, USA), FITC-labeled anti-CD4 (eBioscience CA, USA), and PercCP-eFluor710-labeled anti-CD8 (eBioscience CA, USA) antibodies at 4 °C for 45 min for flow cytometry analysis. APC-labeled anti-OX62 (eBioscience CA, USA) along with PE-labeled anti-CD80 (BioLegend CA, USA) were used to determine the purity of the primary DCs, and APC-labeled anti-CD3 was used for T cells. Cell staining was performed according to the manufacturers’ recommendations. T cell proliferation was measured by bromodeoxyuridine (BrdU) incorporation (APC BrdU Flow Kit, BD Pharmingen NJ, USA) according to the manufacturer’s protocols. Cells stained with APC-labeled anti-BrdU mAb (BD Pharmingen NJ, USA) were analyzed by flow cytometry. Flow cytometry analysis was conducted using a Gallios flow cytometer (Beckman Coulter, USA).