Ifn gamma human uncoated elisa kit

hPBMCs (Lonza, Basel, Switzerland) were cultured in a lymphocyte growth medium (LGM-3, Lonza) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Thermo Fisher Scientific) and 1% penicillin-streptomycin (Thermo Fisher Scientific). Cells were seeded at a density of 1 million cells per well in a 24-well cell culture plate (SPL Life Sciences, Pocheon, Republic of Korea) and treated with ProLNG-001 at the indicated concentration. For cytokine measurement, cells were incubated for 24 h at 37 °C in a humidified 5% CO₂ incubator. After 24 h, the cell culture supernatants were collected, and the IFN-γ secretion was measured using the IFN-gamma Human Uncoated ELISA Kit (Thermo Fisher Scientific). For the analysis of IFN-γ-secreting CD8+ T cells, FACS analysis was performed. The cells were stimulated with a cell stimulation cocktail (plus protein transport inhibitors 0.5×, Thermo Fisher Scientific) for 6 h, after 48 h of incubation with the samples. After stimulation, the cells were intracellularly stained using the Intracellular Fixation & Permeabilization Buffer Set Kit (Thermo Fisher Scientific) following the manufacturer’s recommendations. The cells were stained with APC-Cy7-Live/Dead (Thermo Fisher Scientific), BV421-CD3, PE-CD8, and PerCP-Cy5.5-IFN-γ antibodies (BioLegend, San Diego, CA, USA).

NK cell surface receptor expression and IFNγ release were analyzed after exposure to MV, MV-BiKE, and MV-scFv-Fc-infected tumor cells, or to MV-infected cells with additional vBiKE treatment. For cocultures, 2 × 10⁴ tumor cells were inoculated at MOI 1, the inoculum was replaced by RPMI, 10% FCS 2 h post infection, and 1 × 10⁵ NK cells were added 24 h post infection. Purified vBiKEs (10 ng/µL) and IL-2 (1000 U/mL) were added to designated conditions directly after adding NK cells. Analysis was performed 72 h post infection, i.e., after 48 h coculture. IFNγ concentrations in coculture supernatants were quantified by ELISA using the IFN gamma Human Uncoated ELISA Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. NK cells were labeled with respective antibodies (panel 1: NKG2D, NKp46, DNAM-1; panel 2: NKG2A, KIR2D, NKp44; panel 3: CD69) and surface marker levels were analyzed by flow cytometry.

Cytokine concentrations in supernatants
obtained from T cell cultures were measured with commercially available
kits (IFN gamma Human Uncoated ELISA Kit and IL-2 Human Uncoated ELISA
Kit, both ThermoFisher). Standards and samples were measured in duplicate,
and samples were appropriately diluted to fall within the calibration
curve range. Values of IL-2 and IFNγ were normalized to response
after stimulation with 125 ng/mL soluble αCD3 and αCD28.