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 p…

Materials

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