Ifn γ and tnf α

Concentrations of IFN-γ (No. 201-TA, sensitivity 8 pg/mL), TNF-α (No. DTA00C, sensitivity 5.5 pg/mL), perforin (No. ab46068, sensitivity <20 pg/mL) and granzyme B (No. ab46142, sensitivity <20 pg/mL) in serum and supernatants were measured with sandwich ELISA using a commercial ELISA kit (IFN-γ and TNF-α from R&D, Minneapolis, MN, USA; perforin and granzyme B from Abcam, Cambridge, MA, USA) per manufacturer’s recommended protocol. A standard curve was calculated by plotting optical density versus the log concentration.

Tumor cells were cultured in the presence of MegaFasL at the indicated concentrations as previously described66 (link). FasL (Mega-Fas Ligand^®, kindly provided by Drs. Steven Butcher and Lars Damstrup at Topotarget A/S, Denmark) is a recombinant fusion protein that consists of three human FasL extracellular domains linked to a protein backbone comprising the dimer-forming collagen domain of human adiponectin. The Mega-Fas Ligand was produced as a glycoprotein in mammalian cells using Good Manufacturing Practice compliant process in Topotarget A/S (Copenhagen, Denmark). IFNγ and TNFα proteins were obtained from R&D Systems Inc. (Minneapolis, MN). For tumor cell apoptosis analysis, cells were stained with Alexa Fluor 647 Annexin V (Biolegend) in Annexin V-binding buffer (10 mM Hepes, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) for 30 min at 4 °C. Propidium Iodide was then added to the cell suspension, and cells were analyzed by flow cytometry as previously described67 (link).