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

Overview

This video demonstrates secreted embryonic alkaline phosphatase assay using HEK-Blue interferon-α/β reporter cells to quantify the type-I interferons (IFNs) released following stimulation of plasmacytoid dendritic cells by HIV-1 Infected CD4⁺ T cells.

Protocol

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.

Materials

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