Human ifn γ elisa kits

A tumor was surgically removed from oral cancer patients at UCLA, and oral squamous carcinoma stem cells (OSCSCs) were isolated from those tumors [17 (link),18 (link),19 (link),20 (link)]. OSCSCs and oral tumors isolated from NSG and hu-BLT mice were cultured in RPMI 1640 (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Gemini Bio-Product, West Sacramento, CA, USA). Dr. Nicholas Cacalano (UCLA David Geffen School of Medicine) kindly provided Mia PaCa-2 (MP2) human pancreatic cell lines. MP2 and pancreatic tumors isolated from NSG and hu-BLT mice were cultured in DMEM supplemented with 10% FBS and 2% penicillin-streptomycin (Gemini Bio-Products, West Sacramento, CA, USA). RPMI 1640 supplemented with 10% FBS was used to culture NSG and hu-BLT mice immune cells. Recombinant human IL-2 was purchased from Hoffman La Roche (Nutley, NJ, USA). Human IFN-γ ELISA kits were purchased from Biolegend (San Diego, CA, USA). Phosphate-buffered saline (PBS) and bovine serum albumin (BSA) were purchased from Life Technologies, Carlsbad, CA, USA. Matrigel was purchased from Corning, NY, USA.

Cytokine content (IL-2, IL-17, and IFN-γ) in the supernatants of stimulated/non-stimulated cells was measured by LEGEND MAX™ Human IL-2, Human IL-17A/F, and Human IFN-γ ELISA kits (BioLegend^®, San Diego, CA, USA) according to the manufacturer’s instructions. Absorbance was measured at a 450 nm wavelength using a SpectraMax i3x Multi-Mode Microplate Reader (Molecular Devices LLC, San Jose, CA, USA). The sensitivity of the assay for IL-2, IL-17, and IFN-γ was 4 pg/mL, 2.58 pg/mL, and 5.6 pg/mL, respectively.

The inhibitory effect of QL-1200186 on IL-12/IL-18-induced IFNγ production in NK cells or human whole blood was determined by enzyme-linked immunosorbent assay (ELISA). NK92 cells were stimulated with recombinant human IL-12 (2 ng/mL; 200–12; PeproTech, Cranbury, NJ, USA) and recombinant human IL-18 (5 ng/mL; 9124-IL-050/CF; R&D Systems, Minneapolis, MN, USA) with or without QL-1200186 for 24 h at 37 °C. For human whole blood cells, whole blood (200 μL) was preincubated with the compound for 1 h and then stimulated with recombinant human IL-12 (2 ng/mL) or recombinant human IL-18 (10 ng/mL) for 24 h. After centrifugation, the supernatant was collected, and IFNγ production in the supernatant was measured with human IFNγ ELISA kits according to the manufacturer’s instructions (Biolegend, 430,104).