Ehifng

IFNg measurements were performed as follows: culture medium was collected 48 hours post-transfection and diluted 1:1000 (with additional medium). A human IFNg ELISA kit (Invitrogen, EHIFNG) was used according to the manufacturer's specifications. Briefly, 50 μl of biotinylated antibody reagent was added to each well of a flat-bottom transparent 96-well plate (provided with the kit); 50 μl diluted samples or standards were added in triplicates to separate wells in the plate and incubated for 2 h at room temperature; after three plate washes with Wash Buffer, 100 μl of Streptavidin-HRP solution was added to each well, followed by incubation at room temperature for 30 min and three additional washes; 100 μl of Tetramethylbenzidine (TMB) substrate was added to each well and a color reaction was allowed to develop for 30 min at room temperature in the dark; the reaction was stopped with the addition of 100 μl Stop Reaction Solution to each well. Standards were resuspended in ultrapure water (Thermo, 10977015) and standard curves were obtained from serial dilutions of the standards. IFNg signal intensity was estimated from absorbance readings at 450 nm (on a Tecan M1000 Pro Reader) and final IFNg concentration estimates were obtained by extrapolation from the standard curve.