Cd25 buv395

AIM assays were performed as previously described ^{43 (link)}. Briefly, cryopreserved PBMC were thawed, washed, resuspended in R10, and rested for 3 hours at 37°C. Following the 3-hour rest interval, the appropriate number of cells were transferred to a 48-well plate and subsequently treated with CD40 blocking antibody (Miltenyi Biotec, cat. no. 130–094-133) for a final concentration of 0.5ug/mL for 15 minutes at 37°C. Cells were incubated in the presence or absence of our 10-LRA panel, as previously described. After an 18hr incubation, cells were harvested and stained for 50 minutes at 4°C with the surface staining monoclonal antibodies (mAb); (PD-L1-PE/Cy7 (Biolegend, cat. no. 329717), CD40L-PE (BD Biosciences [BD], cat. no. 561720), OX40-APC (BD cat. no. 563473), CD69-BV650 (Biolegend cat. no. 310933), CD3-BV605 (Biolegend cat. no. 317322), CD4-BV421 (BD cat. no. 562424), CD8-PerCp-Cy5.5 (BD cat. no. 560662), CD25- BUV395 (BD cat. no. 564034) and LIVE/DEAD Near-IR stain (ThermoFisher cat. no. L34975), washed, fixed and permeabilized (BD cytofix fixation buffer, cat. no. 554655), and then stained for 30 minutes at 4°C with intracellular mAb Acetyl-histone H3-Alexa Fluor 488 (Cell Signaling Technology, cat. no. 9683S) in 1x perm/wash buffer (BD, cat. no. 554723). The cells were washed and fixed (BD cytofix fixation buffer, cat. no. 554655) prior to flow cytometry analysis.

After stimulation, PBMCs, monocyte-depleted PBMCs or total leukocytes were washed twice in PBS before centrifugation at 1500 rpm for 5 min (RT) and stained for the following surface markers: CD45 (Alexa fluor 700, Biolegend) CD4 (FITC, Biolegend), CD8 (BV605, Biolegend), CD25 (BUV395, BD), CD69 (APC-Cy7, Biolegend) and live dead (aqua zombie, Biolegend) for 30 min at 4°C in FACS buffer. The staining of TNFR1 and TNFR2 on T cells and monocytes was conducted using the following antibodies: CD4 (FITC, Biolegend), CD8 (BV605, Biolegend), CD14 (BV711, Biolegend), CD16 (BUV735, BD), CD120a (APC, Biolegend), CD120b (PE, Biolegend). Antibodies to CD16 (BUV395, BD), CD11b (Pe-Cy7, Biolegend) and CD62L (APC-Cy7, Biolegend) were used to analyze the phenotype of neutrophils. After staining, the cells were washed twice in FACS buffer and re-suspended in 100 μL/well FACS buffer for analysis. Acquisition was performed using a BD Fortessa coupled to an HTS platform.