Reporter lysis 5x buffer

Cells (7.5×10⁴ cells/well) were seeded in 24-well plates with FBS-free medium at 37°C the day before transfection. Transfection was performed with transfection reagent Fugene-6 (Roche, Basel, Switzerland) according to manufacturer's specifications. NFκB-Luc reporter construct (1 µg/well) (Stratagene, La Jolla, CA, USA) was added to cells along with plasmid CMV-β-galactosidase (CMV-β-gal, 0.2 µg/well; Promega, Madison, WI, USA) and transfection reagent (15 µl/24-well plate). CMV-β-gal served as an internal control to normalize the transfection efficiency. After 24 hr incubation, cell cultures were treated with Tan IIA (1, 3 and 10 µM) or Sal B (200 µM) for 30 min. LPS was then added to stimulate NF-κB activity for 6 hr. Cell lysates were harvested with diluted reporter lysis 5X buffer (Promega). 24-well plates were added reporter lysis buffer (100 µl/well), and 20 µl cell lysate mixed with 100 µl luciferin in white 96-well plates. Luminescence was detected by luminometer-VICTOR2 Multilabel Counter (Perkin Elmer Inc., Waltham, MA, USA), as previously reported by us [27] (link), [28] (link).

To evaluate gene-cargo expression levels for CAR- or TCR-T cells transduced with sense vs antisense lentiviral vectors containing luciferase as the inducible gene cargo under 6xNFAT, 2.5 × 10⁴ UTD and transduced T cells were co-cultured with target tumour cells at 1:1 E:T ratio for 24 h in 96-well plates. The following day, the culture media were washed away and 10 µl per well of opportunely diluted Reporter Lysis 5X buffer (Promega) was added and the cells resuspended. Luciferin (50 µl per well) (PerkinElmer) was then added and cell lysates were transferred into white 96-well white optiplates (PerkinElmer) for bioluminescence acquisition. Luciferase activity was measured by total counts acquired using the HIDEX sense 425-301i plate reader and software (Hidex).

For the reporter assay, A549 or H1299 cells transfected with reporter or mock plasmids were lysed in Reporter Lysis 5X buffer (Promega, Madison, WI, USA). Renilla and firefly luciferase activities were determined by using the Dual-Luciferase^® Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer’s instructions.

Pseudoviruses were titrated and normalized to ensure that the same numbers of virus particles were used for infectivity test before infected into HCC15 receptor stable cells, which are highly susceptible to SARS-CoV-2 infection (unpublished data). Pseudovirus-infected cells were lysed at 72 h post-infection for 5 min on ice with chilled Reporter Lysis 5X Buffer (Promega, USA), and then precipitated by centrifugation (at 15,000 RPM for 15 min at 4°C). 25 ul of each lysate was transferred to a new tube, and mixed with 25ul of Luciferase buffer containing luciferase substrate (Luciferase Assay System, Promega).