Veritas 96 well luminometer

HEK 293T cells were plated at 0.5 × 10⁵ cells/well in 24-well plates (Corning) coated with poly-ornithine (Sigma-Aldrich). The following day, the cells were transfected with DNAs encoding the Envelope protein of interest, an NFAT Firefly Luc (NFAT-FLuc) reporter (using 4× NFAT site from human IL-2 gene), and pRL-TK-Renilla Luc reporter (TK-RLuc, transfection control reporter using HSV TK, herpes simplex virus thymidine kinase, promoter) using Lipofectamine 2000 reagents. The standard transfection ratio of Envelope protein: NFAT-FLuc: TK-RLuc was as follows: 0.3 μg: 0.3 μg: 0.03 μg. Peptides of interest were added to this mixture at an amount of 0.1 μg. The cells were then incubated overnight at 37°C in a CO₂ incubator. The following day, the cells were treated with 1 μM Phorbol 12-myristate 13-acetate (Sigma-Aldrich, P1585) for 8 h at 37°C in a CO₂ incubator. Luc activity levels were then assayed using Dual Luciferase assay kit and Veritas 96-well luminometer (Promega, E1910) following the manufacturer’s instructions. Following the luciferase results, TAT-MY18-2ED peptide (>95% purity) was synthesized by Thermo Fisher/Pierce Custom Peptide team:
TAT-MY18-2ED: GRKKRRQRRRPPQ-MYSFVSDDTGTLIVNSVL (M.W. = 3,662.2416).

HEK 293T cells were plated at 0.5 × 10⁵ cells/well in 24 well plates (Corning) coated with poly-ornithine (Sigma-Aldrich). The following day, the cells were transfected with cDNAs encoding PA-Cre along with Firefly Luc reporter and Renilla Luc using X-treameGENE9 reagent (Sigma-Aldrich). The standard transfection ratio of PA-Cre:double-flox-inverted-FLuc:HSV-TK-Renilla Luc was as follows: 1:9:0.1. The cells were then exposed to blue light (total 12 h, 20 s light/60 s dark cycle, homemade device^{13 (link)}) 36 h after transfection at 37 °C in a CO₂ incubator (Thermo-Fisher). In case of tamoxifen-treated Mock or CreERT2, the cells were treated with 1 μM tamoxifen (Sigma-Aldrich) 36 h after transfection. Luc activity levels were assayed right after illumination was terminated using Dual Luciferase assay kit and Veritas 96-well luminometer (Promega) following the manufacturer’s instructions.