Invitrogen uncoated elisa kits

After harvesting the cells from the stimulation experiments, the supernatants were separated from the cell pellets, collected in round‐bottom 96‐well plates and kept on ice for short‐term storage (< 12 h). To quantify the cytokines IFN‐γ, IL‐2 and TNF, ELISAs were performed using Nunc MaxiSorp™ flat‐bottom 96‐well plates and the respective Invitrogen™ Uncoated ELISA Kits (Thermo Fisher Scientific) according to the manufacturer's protocol. A BioTek ELx405 plate washer was used for washing steps, and absorbance at 450 and 570 nm was measured using a SpectraMax M5 plate reader (Molecular Devices). Standards in duplicates were included for each plate to generate calibration curves for the calculation of cytokine concentrations.

Supernatant cytokine concentration was determined using Invitrogen Uncoated ELISA kits (Thermo-Fisher) for mouse TNF (# 88-7324-88), IL-6 (#88-7064-77), and IL-10 (# 88-7105-88) as per manufacturer's instructions. After the final incubation with streptavidin-horseradish peroxidase conjugates, plates were washed seven times. Fifty μL of TMB substrate reagent was added to each well. The reaction was stopped with 25 μL of 1 M H₂SO₄. The plate was read using a microplate reader set to a wavelength of 450 nm. Microsoft Excel software was used to generate a standard curve from which the cytokine concentration of the samples was determined.