Human ifn γ elisa max deluxe

The culture supernatant was collected from NK cell medium
treated with or without plasma-derived exosomes after 72
hours. The culture supernatant was stored in a -80°C freezer
until assessment of the cytokine. Concentrations of IFN-γ
in the culture supernatant were measured using the Human
IFN-γ ELISA MAX™ Deluxe (Bio-Legend, USA). All
procedures were performed according to the manufacturer’s
instructions.

PBMCs from healthy individuals were stimulated by CpG (10 µg/mL) for 24 h. PMA and ionomycin were then added for the last 4 h of culture and BFA (10 µg/mL, Sigma Aldrich) was added for the last 2 h. PBMCs were then stained using FITC or V450-conjugated anti CD19 (clone HIB19, BD Pharmingen) and Fixable Viability Dye eFluor 506 (Thermofisher) before fixation (cytofix/cytoperm buffer BD biosciences) and permeabilization (Perm wash buffer, BD Pharmingen). Intracellular staining for IL-10, IFNγ, and TNFα was then performed using APC-conjugated anti IL-10 (clone JES3-19F1, BD Pharmingen) and PeCy7-conjugated anti-IFNγ PeCy7 (4SB3, BD Pharmingen) or anti-TNFα (clone MAb11, BD Pharmingen). Percentages of IFNγ and TNFα positive cells among B10⁺ and B10^neg cells were measured by flow cytometry, using FACS CANTO II (BD biosciences). Gating strategy and representative flow cytometry dot plot are shown in Figure S2 in Supplementary Material. We also measured cytokines concentrations in supernatants from isolated B10⁺ and B10^neg cells. To do this, B10⁺ and B10^neg cells were sorted as described above, and kept in culture for 24 h. Then, cytokine dosages in supernatants were performed using Human IFN-γ ELISA MAX Deluxe and Human TNF-α ELISA MAX Deluxe (BioLegend, San Diego, CA, USA) according to the manufacturer’s instructions.