Alkaline phosphatase blue microwell substrate

HEK293T cells were either transiently transfected with luciferase reporter constructs, or reporter cell lines (A549-Dual, 293-Dual hSTING-R232, THP-1-Dual or THP1-Dual KO-cGAS) were used. 32 h post-transfection or 72 h post transduction, cells were lysed in passive lysis buffer (Promega, E1941) and luciferase activities of the firefly luciferase (FFLuc), renilla luciferase, lucia luciferase or SEAP activity were determined^{64 (link)}. For HEK293T cells ISRE-firefly luciferase activities normalised to renilla activity were measured via DualGlo Luciferase Assay System (Promega, E2980). For the reporter cell lines ISRE-lucia luciferase activity (IFNb-lucia luciferase for 293-Dual hSTING-R232 cells) was measured 1 s after injecting 20 mM coelenterazine (PFK Biotech, 102173) and NF-κB-SEAP activity (ISRE-SEAP for 293-Dual hSTING-R232 cells) via Alkaline Phosphatase Blue Microwell Substrate (Sigma-Aldrich, AB0100). Both were normalized to cell viability determined by the CellTiter-Glo Luminescent Cell Viability Assay (Promega, G7570). Luciferase and cell viability measurements were performed using an Orion II microplate Luminometer and the Simplicity software (Berthold), SEAP activity was measured at 650 nm by using a Vmax kinetic microplate reader (Molecular Devices) and the SoftMax Pro 7.0.3 software.

The mesenchymal progenitor cell line C3H10T1/2 was plated in triplicates and grown to full confluency in 96-well plates. Subsequently, cells were exposed to experimental compounds in full growth medium for 4 days. Afterwards, cells were lysed in Passive Lysis Buffer (Promega). A part of the lysate was used for protein quantification (Bio-Rad Protein Assay, Bio-Rad), while the remaining lysate was used for measuring alkaline phosphatase activity (Alkaline Phosphatase Blue Microwell Substrate, Sigma).