Victor x multilabel reader

High-throughput cancer cell viability assays were performed by Reaction Biology Corp. (USA). About 120 major cancer cell lines derived from skin, breast, brain, ovary, liver, stomach, kidney, bone, pancreas, intestine, lung, and blood cancer were inoculated in a 96-well plate at a density of 10⁴ cells/well, and cultured at 37 °C for 24 h, followed by various concentrations (0, 1, 2, 5, 10 µM) of lomitapide (Sigma-Aldrich). The plate was incubated at 5% CO₂ at 37 °C for 24 h after treatment with lomitapide. Thereafter, cells were added to each well of 100 µl of the assay reagent (CellTiter-Glo^® Reagent), and luminescence was measured using a VICTOR X Multilabel Reader (PerkinElmer, USA).

HEK293T cells were transfected with Renilla luciferase vector pGL4.74 hRL-TK (Promega) or plasmids coding for Renilla luciferase-fused PRRSV-N or nsp1α. Transfected cells were lysed 48 h later using passive cell lysis buffer (Promega) supplemented with a protease inhibitor cocktail (Sigma-Aldrich). Cell lysates were further clarified by centrifugation at 13000 rpm for 5 min to remove cell debris then supernatants were transferred to fresh tubes and stored at − 80 °C before use in the LACA assay. Meanwhile, Western blot analysis and an immunofluorescence assay (IFA) were used to test expression levels of recombinant fusion proteins.
Luciferase levels within cell lysates were evaluated using Renilla luciferase substrate (Transgene Biotech, Beijing, China) according to the manufacturer’s instructions and quantified using a VICTORX™ Multilabel Reader (Perkin-Elmer Life and Analytical Sciences, Wellesley, MA, USA). Recombinant Renilla luciferase (Raybiotech, Norcross, GA, USA) was used to determine the relative enzymatic activities of luciferase-fused PRRSV-N or nsp1α from cell lysates. For the LACA, enzyme activity values were assigned to HEK293T cell lysate dilutions in phosphate buffered saline (PBS) (100 μL) based on one enzyme activity equivalent of 1 ng recombinant Renilla luciferase defined as 1 Test Unit (1 TU) unless otherwise specified.