Celltiter 96 aqueous one solution cell proliferation assay mts

Cell viability was assessed by CellTiter 96^® AQueous One Solution (MTS) Cell Proliferation Assay and CellTiter-Glo^® (CTG) Luminescent Cell Viability Assay (Promega, Madison, WI, USA). Cells were seeded in 96-well plates (Nunc) at a density of 60,000 cells per mL, 50 μl per well. On the next day, another 50 μl indicated drugs (concentrations ranging from 0.0001 to 100 μM) were added onto cells per treatment (n = 6) and incubated for 72 h. CellTiter 96^® AQueous One Solution Cell Proliferation Assays and CellTiter-Glo^® Luminescent Cell Viability Assays were conducted following the instructions for use of product. The absorbance at 490 nm or luminescence at 590 nm generated by the cells in each well was recorded with a microplate reader (Tecan, Maennedorf, Switzerland). Each experiment has triplicates. Mean and standard deviation (SD) were calculated. Dimethylsulphoxide (DMSO) treated cells were used as control. Background luminescence (readout of the well with no cell seeded) was subtracted from each sample before calculating the luminescent ratios. The percentage of cell viability of other wells were normalized to the DMSO control. Sigmoidal dose–response curves were generated by fitting calculated cell viability values at different log concentrations while IC₅₀ was calculated using GraphPad Prism (v7.0).

Myoblasts were seeded onto 0.2% gelatin-coated 96-well plates at a density of 2000 cells per well and were stimulated with the indicated recombinant cytokines (IL-15, 25 ng/mL; TNFα, 1 ng/mL in myoblast growth medium). The medium was changed every 48 h and the number of viable cells in culture assessed at days 1, 3, 6 and 9 using the CellTiter 96® Aqueous One Solution MTS Cell Proliferation Assay (Promega) according to the manufacturer’s instructions.

Cytotoxicity of the PY-LCNP-PEG/TEMPO and TEMPO_free was determined using the CellTiter 96^® Aqueous One Solution MTS Cell Proliferation Assay (Promega, Madison WI, USA). Briefly, HeLa cells were seeded to 96-well tissue culture plates (~5 × 10³ cells/well in 100 µL media) and culture for 24 h in standard incubation conditions. Next, 50 µL of DMEM-HEPES containing increasing concentrations of PY-LCNP-PEG/TEMPO or TEMPO_free were added to the wells and incubated for 30 min at 37 °C. After the incubation, the materials were replaced with 100 µL of complete growth medium and the cells were cultured for 72 h. After this proliferation period, 20 µL of the tetrazolium substrate was added to each well and incubated at 37 °C for 3 h for the formation of color product formazan. The absorbance of the formazan product was read at 590 nm and 650 nm (for subtraction of nonspecific background absorbance) using a Tecan Infinite M1000 (Tecan, Morrisville, NC, USA) microtiter plate reader. Absorbance values with the background subtracted were plotted as a function of material concentration and reported as percent of control cell proliferation (cells cultured in complete media only).

The CellTiter 96 Aqueous One Solution (MTS) Cell Proliferation Assay (Promega, Australia) was used to determine cell viability of C2C12 myotubes, according to the manufacturer's instructions. Briefly, C2C12 myotubes stimulated with CSE or H₂O₂ were washed and incubated in media containing MTS reagent at 37°C and 5% CO₂ for 1 h. Absorbance was then recorded at 490 nm using a plate reader (CLARIOstar Monochrome Microplate Reader; BMG Labtech, Australia). To validate the specificity of the assay, MTS reagent was added to unseeded culture plate containing CSE or H₂O₂ for 1 h before absorbance reading. No significant absorbance changes were observed in response to these stimuli verifying the reliability and specificity of the assay.