Cd4 buv395 clone sk3

Human PBMCs from healthy donors were prepared from 50 ml of fresh blood with Ficoll in Leucosep tubes. The tubes were centrifuged at room temperature (30 min, 800 × g) without brake. Isolated cells were washed and staved overnight. The cells were treated with AX-024 or vehicle and incubated for 1 h at 37 °C followed by further incubation on ice. 10, 25, or 50 μg·ml⁻¹ anti-CD3-biotin (clone OKT3, BioLegend) was added to the cells on ice for 15 min before neutravidin was added at a final concentration of 20 μg·ml⁻¹ for cross-linking. After stimulation for 3 min at 37 °C, cells were fixed (BD Cytofix fixation buffer) and permeabilized (TruePhos buffer, BioLegend). Staining was performed with CD3-PerCP (clone SK7, BD), CD4-BUV395 (clone SK3, BD), CD8-BV605 (clone RPAT8, BioLegend), and Zap70-PE (clone 65E4, CST). Flow cytometry and data analysis were done as described above.

For both the in vitro and ex vivo studies PBMC were activated for four hours with CD2/CD3/CD28 T cell activation beads (Miltenyi Biotec, Cologne, Germany) in the presence of GolgiStop and GolgiPlug protein transport inhibitors (BD Bioscience, Franklin Lakes, NJ), and anti-human CD107a APC conjugated antibody (clone H4A3, BD Bioscience). Cell surface marker staining was performed for the in vitro studies using LIVE/DEAD Aqua fixable dead cell stain (Thermo Fisher Scientific), CD4 APC Cy7 (clone RPA-T4, BD Bioscience), CD3 BV785 (clone OKT3, BioLegend, San Diego, CA) CD8α BV650 (clone RPA-T8, BioLegend), and IFNγ PE (clone B27, BD Biosciences). Cell surface marker staining was performed for the ex vivo study using LIVE/DEAD Aqua fixable dead cell stain, CD4 BUV395 (clone SK3, BD Biosciences), CD3 BUV496 (clone UCHT1, BD Biosciences) and CD8α BUV805 (clone SK1, BD Biosciences). Surface and intracellular cytokine staining was performed using the Cytofix/Cytoperm kit (BD Biosciences) according to manufacturer’s instructions.