Elisa max standard set human ifn γ

Peripheral blood mononuclear cells (PBMCs) were stimulated at 1 × 10⁶/ml with a pool containing a maximum of 6 peptides of interest (Peptide2.0; >90% pure) at a concentration of 10 μg/ml/peptide. After 14 days 200,000 cultured PBMCs were restimulated in duplicate for 48 h with each peptide of interest separately or corresponding controls. Interferon γ (IFNγ) production was assessed by subjecting supernatants of restimulation cultures to the ELISA Max Standard Set Human IFNγ (BioLegend, San Diego, USA) according to the manufacturer’s instructions. Absorbance was read at 450 nm using an Infinite 200 PRO microplate reader (Tecan, Männedorf, Switzerland).

For the NKT/PBMC cell assay without APCs, total 5 × 10⁶ PBMCs/well were stimulated with 10 µg/ml αGalCer or 20 µg/ml EhLPPG in 100 µl of RPMI 1640 (10 % FCS, 1 % l-Glutamine, 1 % Pen/Strep) and incubated for 48 h at 37 °C under 5 % CO₂. For the NKT/PBMC cell assay using the resultant APCs, 1 × 10⁵ APCs/well were stimulated with 10 µg/ml αGalCer or 20 µg/ml EhLPPG in 50 µl of RPMI 1640 (10 % FCS, 1 % l-Glutamine, 1 % Pen/Strep) and incubated for 3–4 h at 37 °C under 5 % CO₂. Next, 5 × 10⁶ thawed PBMCs from the negative (CD14⁺ monocyte-depleted) PBMC fraction described above was added in 50 µl of RPMI 1640 to the stimulated APCs, and the mixed samples were incubated for 48 h at 37 °C under 5 % CO₂. Both assays were run in duplicate. After 48 h, supernatants were collected and assayed for IFNγ using ELISA MAX™ Standard SET Human IFNγ (BioLegend).