Elisa kits for ifn γ

Oral squamous carcinoma stem cells (OSCSCs) and oral squamous cell carcinoma (OSCCs) were isolated from patients with tongue tumors at UCLA (13 (link), 42 (link), 55 (link)). NK cells, OSCSCs, and OSCCs were cultured in RPMI 1640 (Invitrogen by Life Technologies, CA), supplemented with 10% fetal bovine serum (FBS) (Gemini Bio-Products, CA). Recombinant IL-2 was obtained from NIH-BRB. Antibodies to CD16 were purchased from Biolegend (San Diego, CA). Antibodies used for flow cytometry – IgG2, MHC-class I, and B7H1 (PD-L1) were purchased from Biolegend (San Diego, CA). MIA PaCA-2 (MP2), PL12, and Capan human pancreatic cancer cell lines were provided by Dr. Nicholas Cacalano (UCLA, School of Medicine, CA, USA), and were cultured in DMEM supplemented with 10% FBS. Cisplatin and Paclitaxel were purchased from Ronald Reagan Pharmacy at UCLA. ELISA kits for IFN-γ were purchased from Biolegend (San Diego, CA), and multiplex analysis kit was purchased from Millipore (Billerica, MA). Propidium iodide (PI) and chromium-51 was purchased from PeproTech (Cranbury, NJ, USA) and Perken Elmer (Waltham, MA, USA), respectively. Chromium Single cell 3’ Reagent kit v3, Cat#1000075 was purchased from10X Genomics (Pleasanton, CA, USA).

6 × 10⁵ splenocytes or 3 × 10⁵ lymph node cells were isolated from mice at day 10 after immunization and seeded in 96 well-plates. The cells were incubated for 72 h in DMEM medium with 5% FCS in the presence of 20 µg/ml of MOG_35–55 peptide. Cytokine levels in the culture medium were determined using commercially available ELISA kits for IFNγ, TNFα, IL-17A (BioLegend), or GM-CSF (R&D Systems, Wiesbaden, Germany) according to the manufacturer’s instructions.