Muse count and viability assay kit

Cell cytotoxicity was estimated by measuring both the cell metabolic activity (MTT assay) and the membrane integrity test (The Muse^TM Count and Viability Kit, Millipore). Cells were seeded in 96-well plates at a density of 2 × 10⁶ cells/well, grown for 12 h and then treated with inhibitors at the indicated concentrations for 1 h at 4 °C. After two washes with M199 medium, cell viability was determined using CCK-8 (Dojindo Molecular Technologies, USA) according to the manufacturer’s instructions. Untreated cells were used as the controls. After adding the CCK-8 solution, absorbance at 450 nm was measured using a plate-reading luminometer (GloMax®-Multi+ Detection System). Cells were grown in a 24-well plate format and treated with inhibitors at the indicated concentrations for 1 h at 4 °C, before assaying with a Muse Cell Analyzer (Merck Millipore, USA). Floating and adherent cells were collected after the various treatments. Cells were washed twice with PBS and resuspended in PBS for a cell viability assay. The cell viability was ascertained with the Muse Count and Viability Assay Kit (Merck Millipore, USA). The calculations were performed automatically, and the viability profiles (dot plots) were displayed using the Muse™ Count and Viability Software Module. The experiment was carried out in triplicate, and the error bars represent the standard deviation.

Human embryonic kidney (HEK) 293T and IMR90 female human fetal lung fibroblast cells were obtained from American Type Culture Collection. All cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific) and 1% antibiotic-antimycotic solution (Thermo Fisher Scientific). IMR90 cells were kept at 5% CO₂ and ambient O₂ at 37°C. All cell lines were regularly tested for mycoplasma contamination using the MycoAlert Mycoplasma Detection Kit (Lonza). Cell counting and viability were performed using the Muse Count and Viability Assay Kit in a Muse Cell Analyzer (Merck Millipore).

Analysis of cell proliferation rate was performed as in Woods et al., 2017 (link). In brief, 10,000 vector control and Bap1 KO mTSCs were plated in complete medium and collected every 24 hr over 4 days. After trypsinization, the number of viable cells was counted using the Muse Count and Viability Assay Kit (Merck Millipore MCH100102) and run on the Muse cell analyser (Merck Millipore), according to manufacturer’s instructions. Statistical analysis was performed using ANOVA followed by Holm-Sidak’s post hoc test.