Quantification of Bioactive Type-I Interferons using a Secreted Embryonic Alkaline Phosphatase Reporter Assay

To measure bioactive type-I interferon using HEK-Blue interferon-alpha/beta reporter cells, first, plate the reporter cells at a density of 50,000 cells per well in a 96-well flat bottom plate to a final volume of 180 microliters. Then, add 20 microliters of the just harvested co-culture supernatant to each well in duplicate. Include a set of internal standard controls, and incubate the plate at 37 degrees Celsius and 5% carbon dioxide for 18 to 22 hours.

To determine the levels of alkaline phosphatase released from the reporter cells, add 180 microliters of QUANTI-Blue solution to each well of a new flat bottom plate. Then, add 20 microliters of the induced HEK-blue interferon-alpha/beta cells supernatants to each well, and incubate the plate at 37 degrees Celsius until color develops in the standard interferon control wells.

The QUANTI-Blue solution changes from pink to purple-blue in the presence of the enzyme. Finally, use a spectrophotometer to evaluate the levels of secreted alkaline phosphatase at 620 to 655 nanometers and determine the concentration of type-I interferon by extrapolation from the linear part of the interferon standard curve.