Celltiter 96 aqueous non radioactive mts assay

Cell proliferation rates of rat adipose-derived and bone-marrow-derived mesenchymal stem cells were assessed at 1, 3, 5, and 7 days of culture using the CellTiter 96 Aqueous Non-Radioactive (MTS) assay (Promega, Madison, WI, USA) as previously described [46 (link),47 (link)]. Briefly, all the experiments were performed using 24-well plates with 1.0 × 10⁴ rBMSC or rAdMSC (passage 5 and passage 6, respectively) seeded in 500 µL of complete growth medium as the seeding volume. The optical density of the cell and MTS reagent complex was measured using a microplate fluorescence reader (BioTek, Winooski, VT, USA) at 490 nm. Medium without cells was used as a blank. Each timepoint was performed in triplicate, and an average absorbance at 490 nm (corrected by the reference blank reading) of the three wells was utilized as data for further analysis. The absorbance data was analyzed for the time effect and coefficients of determination (R² values) of the linear regression were calculated for a direct comparison. At each timepoint, an additional well was utilized for qualitative cell viability analysis using Calcein AM Viability Dye (Thermo Fisher, Waltham, MA, USA); stained cells were visualized after 5 min, and images were obtained using a fluorescent microscope (Leica Microsystems, Wetzlar, Germany).

Low passage (P2–P4) equine MSCs were seeded at a density of 2 × 10⁴ per well in a 24-well tissue culture plate. The medium was replaced every 2-3 days. Cell proliferation was measured at 2, 4, and 7 days after seeding. A CellTiter 96 Aqueous nonradioactive (MTS) assay (Promega, Madison, WI) was used following the manufacturer instructions. Briefly, MTS reagent in a 5 : 1 ratio relative to the media was added to each well and incubated for 3 h at 37°C and 5% CO₂. The absorbance at 490 nm was measured and data was obtained using Gen5 data analysis software (Biotek, Winooski, Vermont). Growth medium only with no cells was used as a blank to correct the readings for each of the samples.

The cellular viability and proliferation of 2.0 × 10⁴ cells of P2–P3 of expanded equine BMMSCs and SFMSCs from each donor (n = 5) was assessed at 2, 4, and 8 days using the CellTiter 96 Aqueous Non-Radioactive (MTS) assay (Promega, Madison, WI, USA), as described previously (20 (link), 22 (link)). The optical density of the cell and the MTS reagent complex was measured on a microplate fluorescence reader (BioTek, Winooski, VT, USA) at 490 nm. Medium without cells was used as a blank. Graph of absorbance at 490 nm vs. days of proliferation was generated.