Celltiter 96 aquous one solution cell proliferation assay mts assay

The cells were plated on 96-well plates at a density of 1.0 × 10⁴ cells/well for cell proliferation assay (MTS assay) or 1.0 × 10³ cells/well for the measurement of cell population doubling level in standard medium and cultured at 37 °C in a humidified 5% CO₂ atmosphere. The culture medium was changed every other day. The proliferation rate of the cells on six 96-well plates was determined on days 1, 2, and 3 using a CellTiter 96^®AQuous One Solution Cell Proliferation Assay (MTS assay) (Promega Corporation, Madison, WI, USA).

DsRed-PPU7 cells were plated on a 96-well plate at a density of 1250 to 20,000 or 2000 cells/well and cultured in the standard medium for 24 h. Then, the medium was changed to a fresh standard medium and cells were cultured for 6 days at 37 °C in a humidified 5% CO₂ atmosphere; the standard medium was changed every other day. DsRed-PPU7 cells were photographed with an exposure time of 1/8 seconds using an inverted fluorescence microscope (Biozero BZ-8100, Keyence, Osaka, Japan) and fluorescence intensity was analyzed using Image J software. Subsequently, the proliferation rate against the same cells was determined with a CellTiter 96^®AQuous One Solution Cell Proliferation Assay (MTS assay; Promega Corporation, Madison, WI, USA). The coefficient of determination (R²) was calculated from a regression line prepared on the basis of fluorescence intensity or the cell proliferation rate against cell density or cell-culture days.

The cells were plated on 96-well plates (n = 6) at a density of 1500 cells/well in standard medium, and cultured at 37 °C in a humidified 5% CO₂ atmosphere. The culture medium was changed every other day. The proliferation rate of the cells on six 96-well plates was determined on days 1, 2, 3, and 5 using a CellTiter 96^®AQuous One Solution Cell Proliferation Assay (MTS assay) (Promega Corporation, Madison, WI, USA).