Bright glo luciferase assay buffer

293T cells were seeded on 10-cm-diameter dishes, cultured for 24 h at 37°C with 5% CO₂, and transfected using polyethyleneimine (Sigma-Aldrich, Poland) with the lentiviral packaging plasmid (psPAX), the VSV-G envelope plasmid (pMD2G), or SARS-CoV-2 S glycoprotein (pCAGGS-SARS-CoV-2-S) and a third plasmid encoding firefly luciferase protein (pRRL luciferase). Cells were further cultured for 72 h at 37°C with 5% CO₂, and pseudoviruses were collected every 24 h and stored at 4°C.
A549^ACE2+ cells were seeded in 48-well plates, cultured for 24 h at 37°C with 5% CO₂, and transduced with pseudoviruses harboring VSV-G or S-SARS-CoV-2 proteins or lacking the fusion protein (ΔEnv) in the presence of Polybrene (Sigma-Aldrich, Poland) (4 μg/ml) and HTCC-77 (100 μg/ml) or control PBS. After 4 h of incubation at 37°C, unbound virions were removed by three washes with 1× PBS, and cells were further cultured for 72 h at 37°C with 5% CO_2. Cells were lysed in Bright-Glo luciferase assay buffer (Promega, Poland) and transferred onto white 96-well plates. Luminescence levels were measured on a Gemini EM microplate reader (Molecular Devices, United Kingdom).