Quanti luc luciferase substrate

THP-1-dual reporter cells (InvivoGen) were stably integrated with two inducible reporter constructs, allowing the activation of the NF-kB or IRF pathways to be detected via measurement of secreted alkaline phosphatase or luciferase activity, respectively. At different time points post poly (dA:dT), poly I:C (4 μg/mL), or virus stimulation, 20 μL aliquots of media were sampled into 96-well plates. One hundred μL of Quanti-Luc luciferase substrate (InvivoGen) were added to each well, and plates were read immediately for luciferase activity using a GloMax 96 Microplate Luminometer (Promega).

In order to measure anti-CD19 TriloBiTE-mediated NFAT activation responses, 10⁵ Raji target cells were seeded in 96-well U-bottom plates. Subsequently, 10⁵ Jurkat NFAT-Lucia reporter cells (Invivogen), were added to each well. Purified TriloBiTEs (diluted in PBS) were added in 3-fold serial dilution, starting from 1 nM. Phorbol myristate acetate (PMA)/ionomycin was included as a positive response control. After 24 h of co-culture, the supernatants were collected and mixed with an equal volume of QUANTI-Luc luciferase substrate (Invivogen, Cat# rep-qlc-1). Luminescence was measured immediately using a BioTec H1MFG Synergy plate reader.
For the assessment of CAR-induced ITAM-signaling, Jurkat-NFAT-mCherry reporter cells (kindly provided by Melita Irving, Ludwig Cancer Research Lausanne) were transduced with CAR-GFP constructs as described above. 10-14 days after transduction, 10⁶ CAR-GFP transduced Jurkat reporters were seeded in 24-well plates together with 10⁶ target cells. After 24 h of co-culture, Jurkat-NFAT-mCherry cells were harvested by pipetting, washed in FACS buffer (5% FBS, PBS) and analyzed for GFP and mCherry expression by flow cytometry.