Monoclonal anti human cd3 apc antibody clone ucht1

After isolation by Ficoll, 2 × 10⁵ human peripheral blood cells (PBMCs) were co-cultured with 1 × 10⁵ or 2 × 10⁴ CCs, ASCs, and BMSCs, plated 2 days before. After 4 days of co-culture, PBMCs were collected and stained with monoclonal anti-human CD3-APC antibody (Clone UCHT1, Biolegend, San Diego, CA, USA) for gaiting lymphocytes and with monoclonal anti-human CD4-PE/Cy7 antibody (Clone RPA-T4, Biolegend, San Diego, CA, USA), and monoclonal anti-human CD8-PerCP antibody (Clone SK1, Biolegend, San Diego, CA, USA) to evaluate the ability of cells to modify the CD4⁺/CD8⁺ T cells ratio. Cells were analyzed on a Cytoflex flow cytometer acquiring 10,000 events.

After isolation by Ficoll, 2×10⁵ human peripheral blood cells (PBMCs) were directly co-cultured in DMEM-based medium with 1×10⁵ ASCs, previously primed or not primed for 48 h with 1 ng/ml of IL-1β. Non co-cultured PBMCs were used as control. After 4 days of co-culture, PBMCs were collected and stained with monoclonal anti-human CD3-APC antibody (Clone UCHT1, Biolegend) for gating lymphocytes. PBMCs were also stained with monoclonal anti-human CD4-PE/Cy7 antibody (Clone RPA-T4, Biolegend) and monoclonal anti-human CD8-PerCP antibody (Clone SK1, Biolegend) to evaluate the ability of ASCs to modify the CD4⁺/CD8⁺ T cells ratio. Cells were analyzed on a Cytoflex flow cytometer acquiring a minimum of 10,000 events.