Muse count and viability kit

Cell viability was assayed with Muse Count and Viability Kit (Millipore, Hayward, CA, USA), which differentially stains viable and dead cells based on their permeability to two DNA binding dyes. The samples were measured on the Muse Cell Analyzer (Millipore) with MuseSoft 1.4.0.0 software.

Cell viability was performed by Muse Count and Viability Kit (Millipore, Hayward, CA, USA) following the manufacturer's protocols. This assay based on differentially staining of viable and dead cells based on their permeability to two DNA binding dyes. The viability was determined by Muse™ Cell Analyzer (Millipore).

Cells were seeded in triplicate in 96-well plates at a density of 7,000 cells/well. 24 hours later, cells were treated with or without various concentrations of RCE for different durations. Cell viability was measured with the Cell cytotoxicity assay kit (Abcam) according to the manufacturer’s instructioms. The results are representative of an average of at least 4 independent experiments. Data were presented as proportional viability (%) by comparing the treated group with the untreated cells, the viability of which is assumed to be 100%.
Cell viability was also measured with the Muse™ Cell Analyzer (Millipore, Hayward, CA, USA) using the Muse Count and Viability Kit (Millipore, Hayward, CA, USA) which differentially stains viable and dead cells based on their permeability to two DNA binding dyes. Briefly, cells were plated onto 12-well plates (50 × 10⁴ cells/ well). The day of treatment cells were counted to estimate the approximate number of cells per well. Following RCE treatment at indicated times, viable cells were counted using Muse™ Cell Analyzer.

Viability of the cells following treatment was determined using the Muse™ Count and Viability kit (Muse™Cell Analyzer; Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. Briefly, cells were seeded in triplicate in 6-well plates at a density of 1×10⁴cells/well. After 24 h incubation, cells were exposed to increasing concentrations of trabectedin (0.1, 1, 10, 100 nM). Then, plates were incubated at 37^°C in a 5% CO₂ incubator for 24, 48, and 72 h. After incubation, all cells were collected and diluted with phosphate buffered saline (PBS). 50 μl of cell suspension was then added into 450 μl MUSE Count and Viability reagent (10x dilution), incubated for 5 min at room temperature, and analyzed using the MUSE Cell Analyzer. Data were presented as proportional viability (%) by comparing the trabectedin treated group with the untreated cells, the viability of which is assumed to be 100%.

The viability of MCF-7/WT and MCF-7/DOX-1 cells (biological duplicates) at a density of 8x10⁴ cells/well were measured via the Muse Cell Analyzer (Millipore, Hayward, CA, USA) using the Muse™ Count and Viability Kit (Millipore, Hayward, CA, USA) after 24 h. Total volume of the cells was 1 mL.

Culture density was assessed in cells plated in 24-well plates and treated the following day with indicated treatments or vehicle (ethanol for 1,25D3; methanol for 4MU; H₂O or PBS for DON). After 96 hours, cells were fixed with 1% glutaraldehyde in PBS, stained with 0.1% crystal violet and air-dried overnight. The following day, stain was resuspended in 0.2% Triton X-100 and absorbance was read at 590 nm as an indicator of adherent cell density. Treatments included 1,25D3, 4MU, DON or vehicles as indicated in figure legends (all from Sigma). Low (29 kDa) and High (1.54 × 10⁶ kDa) molecular weight HA (R&D Systems, Minneapolis, MN) were suspended in H₂O and added to media at a final concentration of 500 μg/mL. Cell cycle, viability and annexin positivity was assessed on a Millipore MUSE cell analyzer using Muse^® Count and Viability kit, Cell Cycle kit and Annexin V & Dead Cell Kit according to manufacturer’s directions.

Cells were seeded in triplicate in 96-well plates at a density of 5,000 cells/well. After 24 h of culture, cells were treated with or without various concentrations of Rhus coriaria extract for different durations. Cell viability was measured with the Cell Cytotoxicity Assay Kit (Abcam) according to the manufacturer’s specifications. The results are representative of an average of 5 independent experiments. Data were presented as proportional viability (%) by comparing the treated group with the untreated cells, the viability of which is assumed to be 100%.
Cell viability was also measured using the Muse™ Cell Analyzer (Millipore, Hayward, CA, USA) using the Muse Count and Viability Kit (Millipore, Hayward, CA, USA) which differentially stains viable and dead cells based on their permeability to two DNA binding dyes. Briefly, cells were plated onto 12-well plates (50 × 10⁴ cells/well) and allowed to grow for 24 h. The day of treatment cells were counted to estimate the approximate number of cells per well. Following RCE treatment at indicated times, viable cells were counted using Muse™ Cell Analyzer.