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Assay Method Information

Assay Name:  STING Human Cell Reporter Assay
Description:  Agonism of the human STING pathway is assessed in THP1-ISG cells (Invivogen, cat #thp-isg) derived from human THP1 monocyte cell line by stable integration of an interferon regulatory factor (IRF)-inducible SEAP reporter construct. THP1-Blue ISG cells express a secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of an ISG54 minimal promoter in conjunction with five interferon (IFN)-stimulated response elements. As a result, THP1-Blue ISG cells allow the monitoring of IRF activation by determining the activity of SEAP. The levels of IRF-induced SEAP in the cell culture supernatant are readily assessed with alkaline phosphatase detection medium, a SEAP detection reagent. These cells are resistant to Zeocin. 2′3′cGAMP was used as a positive control in this assay. To run the assay, 60,000 cells were dispensed in 30 μL/well of a white, opaque bottom tissue culture treated 384-well plate.Test compounds were added in a volume of 10 μL (1% DMSO final concentration). Compounds are initially prepared in 100% DMSO, spotted on an intermediate dilution plate and then diluted in media prior to transfer. The assay was incubated for 24 h at 37 C., 5% CO2 then plates were centrifuged at 1200 rpm (120g) for 5 min. After final incubation, 90 μL of alkaline phosphatase detection medium-substrate was added to each well of a new 384-well clear plate and 10 μL of the cell supernatant was transferred from the assay plate to the new alkaline phosphatase detection medium-plate using a Biomek FX and mixed 4 times. Plates were incubated at RT for 20 min then absorbance at 655 nm was determined on the Tecan Safire2.
Affinity data for this assay
 

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Last update November 1, 2007
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