Cd4 pe

Ex vivo PBMCs were immediately thawed, washed in cold wash buffer (phosphate-buffered saline, 2% fetal bovine serum, and 1 mM ethylenediamine tetraacetic acid). Cells were blocked with wash buffer containing 5% of heat-inactivated human plasma, followed by staining with CD16-FITC (SouthernBiotech), TRAIL-PE or isotype-matched antibody (BD Biosciences), CD3− Pacific Blue (BioLegend), CD107a-eFluor660 or isotype-matched antibody, and CD56-PE.Cy5.5 (eBioscience), and fixed with 2%paraformaldehyde prior to analysis. Cell cultures were stained with the same antibodies as patients' sample but CD56-PerCP.Cy5.5 (BioLegend) was used instead. For TLR3 detection, cells were further permeabilized with BD®Perm reagent and stained with TLR3-PE (BD Pharmingen). PDCs were assessed with the following antibody cocktail: CD4-PE (SouthernBiotech), CD11c-PE.Cy7 (BD Pharmingen), and BDCA4-APC (Miltenyi). About 1 × 10⁵ events in the lymphocyte gate were acquired when analyzing patients' samples in FACS Aria IIu. For in vitro samples, 5 × 10⁵ events were acquired in the same gate using BD Accuri C6 or FACS AriaIIu. All samples were analyzed in up to 18 hours after fixation. IL12 (R&D Systems) and IFNα (PBL Interferon Source) were detected in cell culture supernatant or plasma samples by ELISA.

Lymphocytes were isolated from bursal samples and were used to study T-cell infiltration by flow cytometry as previously described (Carballeda et al., 2011 (link)). Briefly, bursas were mechanically disrupted in RPMI 1640 and cellular suspensions were passed through a 40 μm mesh (Cell Strainer, BD). Mononuclear cells were isolated by centrifugation over a Histopaque density gradient. About 1 × 10⁶ cells per well were seeded on a 96-well plate and stained with different combinations of antibodies. Monoclonal antibodies (mAbs) (CD3-SPRD, CD4-PE, CD8α-FITC, Bu-PE) were purchased from SouthernBiotech (Birmingham, AL, United States). Cell suspensions were analyzed with a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, United States) and CellQuest software. The lymphocyte gate was defined by the forward/side scatter characteristics of the cells and 50,000 events were analyzed for each sample. Individual values of all experimental groups were normalized to the mean values of unchallenged healthy group.