Anti cd4 pe a161a1

CD19⁺ B lymphocytes and CD4⁺ CD25^−/int CD127⁺ conventional T-helper (Tcon) lymphocytes were isolated from peripheral blood of melanoma patients using the BD FACS Aria II cell sorter. Purified B and Tcon lymphocyte suspensions were co-cultured (1x10⁵ each/well) in sterile DMEM (10% FBS, 50U/ml Pen-Strep) media containing Dynabeads® Human T-Activator CD3/CD28 (1x10⁵ beads/well) and 10 U/ml recombinant IL-2. Tcon cells (1x10⁵) were also cultured alone as control. 100µl per well was added to round-bottom 96 well plates for each condition. The plates were incubated at 37°C with 5% CO2 for 72 hours.
Post-culture, cells were washed twice and LIVE/DEAD Near-IR Fixable dye was added. Cells were then incubated with Human Fc block (BD Biosciences) prior to extracellular labeling with anti-CD4-PE (A161A1, BioLegend). Cells were washed and fixed with Foxp3 Fixation/Permeabilization Solution (Thermo Fisher Scientific). The cells were then washed in Permeabilization Solution (Thermo Fisher Scientific) and intracellular labeling performed with anti-FOXP3-AF488 antibody (259D, BioLegend). Cells were washed, acquired on the CytoFLEX Flow Cytometer (Beckman Coulter) and analyzed in FlowJo v10.4.

CD19⁺ B lymphocytes and CD4⁺ T-helper (Th) lymphocytes were isolated from peripheral blood of melanoma patients using RosetteSep™ Human B and CD4⁺ T Cell Enrichment Cocktails, respectively (STEMCELL Technologies). Purified B and Th lymphocyte suspensions were stained with 0.5µM eBioscience Cell Proliferation Dye eFluor 670 (Thermo Fisher Scientific) in PBS for 10 minutes at 37°C and washed three times in sterile ExCellerate B Cell Media (Bio-Techne) with 50U/ml Pen-Strep. B and T lymphocytes were co-cultured (1x10⁵ each/well) in media containing Dynabeads® Human T-Activator CD3/CD28 (1x10⁵ beads/well) and 10 U/ml recombinant IL-2. 50µg/ml Nivolumab (Bristol Myers Squibb) was added to selected co-culture wells. Th cells (1x10⁵) were also cultured alone as control. 0.01–1 µg/ml recombinant IL-10 or TNF-α (BioLegend) was added to selected T-helper monoculture wells. 100µl per well was added to round-bottom 96 well plates for each condition. The plates were incubated at 37°C with 5% CO₂ for 72 hours.
Post-culture, cells were washed twice and LIVE/DEAD Near-IR Fixable dye was added. Cells were then incubated with Human Fc block (BD Biosciences) prior to extracellular labeling with anti-CD4-PE (A161A1, BioLegend). Cells were washed, acquired on the CytoFLEX Flow Cytometer (Beckman Coulter), and proliferation modeling was performed in FlowJo v10.4.