Pe labeled anti cd80

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).

Immature DC, mDC-LPS, mDC-Pam₃CSK₄ and, iDC treated with PGE₂ were harvested by gentle flushing with 1 mL of culture medium, centrifuged for 5 min at 340 g, incubated in staining buffer (PBS, 2 mM EDTA, 0.5% FBS) with a 1:100 dilution of Human TruStain FcX (Biolegend) for 5 min at room temperature. Cells were then stained in cold staining buffer for 25 min with fluorophore-conjugated antibodies (APC-Cy7 labeled anti-HLA-DR (clone: L243, 1/400), PE labeled anti-CD80 (clone: 2D10, 1/200), PerCP-Cy5.5 labeled anti-CD86 (clone: IT2.2, 1/400), Pacific Blue labeled anti-PD-L1 (clone: 29E.2A3, 1/400), PerCP-Cy5.5 labeled anti-CCR5 (clone: J418J1, 1/400), and PE labeled anti-CCR7 (clone: G043H7, 1/400)) from Biolegend. After staining, the cells were washed with cold staining buffer, centrifuged twice for 5 min at 340 g at 4°C, and analyzed by flow cytometry using LSR-II flow cytometer (BD Biosciences) and the associated BD FACSDiva software. Data were then analyzed using the FlowJo 7.6.5 software (TreeStar).