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

Abstract

Source: Bego, M. G. et al., Assessing the Innate Sensing of HIV-1 Infected CD4⁺ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System. J. Vis. Exp. (2015)

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 ....

Materials

List of materials used in this article

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