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