Apc anti human ifn γ

Splenocytes and PBMCs were prepared from mice and NHPs respectively. Splenocytes or PBMCs (1 × 10⁶) were seeded into 96-well round bottom plates (Corning, Cat. 3799) and stimulated with 1% DMSO as negative control, 1 μg/mL/peptide of a peptide pool from SARS-CoV-2 spike protein (synthesized by GenScript, China) or 100 ng/mL PMA & 1 μg/mL Ionomycin as positive control. 1 μg/mL Brefeldin A (BD, Cat. 555029) was added to inhibit cytokine secretion. After incubation at 37 °C 5% CO₂ for approximately 6 h, cells were harvested and stained with LIVE/DEAD Aqua and a panel of flow cytometry antibodies specific for cell surface markers: PB anti-mouse CD3 (BioLegend, Cat. 100214), FITC anti-mouse CD4 (BD, Cat. 553047) for mouse study or PB anti-human CD3 (BD, Cat. 558124), FITC anti-human CD4 (BD, Cat. 550628) for NHP study at 2–8 °C for 30 min. Following washing and permeabilization (BD, Cat. 554714), cells were further stained with flow cytometry antibody mixture: APC anti-mouse IFN-γ (BD, Cat. 554413), PE-Cy7 anti-mouse IL-2 (BD, Cat. 560538), PE anti-mouse TNF-α (BD, Cat. 554419) for mouse study or APC anti-human IFN-γ (BD, Cat. 554702), PE-Cy7 anti- human IL-2 (BD, Cat. 560707), PE anti- human TNF-α (BD, Cat. 557068) for NHP study at 2–8 °C for 30 min. The stained cells were analyzed by BD FACSCantoII flow cytometry.