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

Protokol

1. Measurement of bioactive type-I IFN using HEK-Blue IFN-α/β reporter cells.

Note: These cells were generated by stable transfection of HEK293 cells with the human STAT2 and IRF9 genes to obtain a fully active type I IFN signaling pathway. They also contain the secreted alkaline phosphatase (SEAP) reporter gene under the control of the IFN-α/β inducible ISG54 promoter. Stimulation of these cells with type-I IFN activates the JAK/STAT/ISGF3 pathway and induces the production of SEAP.

1. Maintain HEK-Blue IFN-α/β cells in DMEM supplemented with 10% FBS. Plate them at a density of 50,000 cells per well in a flat-bottom 96-well plate, with a final volume of 180 µl.
2. Mix 220 µl of PBMCs (diluted at 850,000 cells/ml) or 220 µl of plasmacytoid dendritic cells (pDCs) (at a concentration ranging from 100,000 to 500,000 cells/ml) with 30 µl of infected T cells (diluted at 10⁶ cells/ml) per well in a U-bottom 96 well plate.
3. Incubate co-cultures at 37 °C and 5% CO₂ for 18-22 hr, and then transfer them to a V-bottom 96 well plate. Centrifuge for 5 min at 400 x g.
4. Add 20 µl of co-culture supernatant to each well in duplicate. Each plate also requires a set of internal standard controls (human IFNα, final concentration from 100 U/ml to 2,500 U/ml). Incubate the plates at 37 °C and 5% CO₂ for 18-22 hr.
5. Determine levels of alkaline phosphatase activity using QUANTI-Blue solution, which changes from pink to purple/blue in the presence of the enzyme. Prepare QUANTI-Blue following the manufacturer's recommendations. Add 180 µl of this solution to each well of a flat-bottom 96-well plate. To each well also add 20 µl of the induced HEK-Blue IFN-α/β cells supernatants and incubate the plate in a 37 °C incubator until color develops in the standard IFN control wells.
6. Evaluate levels of SEAP using a spectrophotometer at 620-655 nm and determine the concentration of type-I IFN by extrapolation from the linear part of the IFN standard curve.

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Malzemeler

Name	Company	Catalog Number	Comments
MT4 cells	NIH AIDS Reagent Program	120	This reagent was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: MT-4 from Dr. Douglas Richman.
HEK-Blu IFN-α/β reporter cells	Invivogen	HKB-IFNAB
RPMI	Wisent	350-000-CL
DMEM	Wisent	319-005-CL
FBS	Wisent	080-150
PHA-L	Sigma-Aldrich	O2769
CD4+ T cell isolation kit II	Myltenyi	130-091-155
Diamond Plasmacytoid Dendritic Cell Isolation Kit II	Myltenyi	130-097-240
QUANTI-Blue	Cedarlane	REP-QB2
2.5 mg/ml human IgG	Sigma-Aldrich	I 4506