Nano glo luciferase assay buffer

HEK293T cells were seeded in 24- or 96-well plates and allowed to attach overnight in full medium. The following day, cells were transfected with 1 μg ml⁻¹ plasmid DNA (35% pAdTrack-CMV, 30% pMSCV-puro-CD14, 30% pMSCV-neo-UNC93B1 and 5% pNL3.2.NF-κB-RE) using Lipofectamine 3000. After 3 h, transfection medium was carefully aspirated and replaced with fresh media for 24 h. The following day, treatments were performed as indicated for 20–24 h, at which time media was removed and a passive lysis buffer (Nano-Glo Luciferase Assay Buffer; Promega) was added, and lysates were incubated on a horizontal shaker for 10 min at 25 °C with 500 reads per million total reads (RPM). Lysates were then transferred to an opaque 96-well plate for measurement of GFP (co-expressed with gene-of-interest (GOI) from pAdTrack-CMV) fluorescence as an indicator of cell density and transfection efficiency on a Synergy Mx plate reader. Subsequently, the NanoLuc substrate Furimazine was diluted in lysis buffer, added to lysates with a multichannel pipette and incubated with agitation for 2 min at room temperature. Luminescence was then quantified using a Synergy Mx plate reader. Luciferase activity was calculated as luminescence divided by GFP fluorescence and reported as a relative fold change (FC).