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

Published: September 29, 2023

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…

Divulgazioni

The authors have nothing to disclose.

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

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Citazione di questo articolo
Quantification of Bioactive Type-I Interferons using a Secreted Embryonic Alkaline Phosphatase Reporter Assay. J. Vis. Exp. (Pending Publication), e21699, doi: (2023).

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