Celltiter 96 aqueous one cell proliferation assay reagent

NIH3T3 cells were seeded in 96-well plates at 2500 cells/well and were transfected 24 h after seeding with 200 ng of each pTargeT^TM construct in triplicates. Transfected cells were maintained in DMEM media supplemented with 2.5% NBCS. At 48 and 72 h post-transfection, the number of metabolically active cells per setup was measured after the addition of 10 µL CellTiter 96^® Aqueous One-Cell Proliferation Assay Reagent (Promega) per well, and incubation at 37 °C until color development. Absorbance values of each setup were measured at 460 nm with a colorimetric plate reader (FLUOstar Omega Microplate Reader, BMG LABTECH, Cary, NC, USA). Cell counts were calculated from a standard curve generated using serial dilutions of an untransfected cell suspension and by plotting the number of cells vs. A₄₆₀. Mean cell counts were calculated per setup for each time point.

In 96-well plates, NIH3T3 cells were seeded at 2500 cells/well. At 24 h post-seeding, cells were transfected with 200 ng of each pTargeT^TM construct in triplicate, after which they were maintained in DMEM media supplemented with 10% NBCS. At 48 and 72 h post-transfection, 10 µL of CellTiter 96^® Aqueous One-Cell Proliferation Assay Reagent (Promega) was added to each well and incubated at 37 °C for 1 h. For each setup, absorbance values were measured at λ = 460 nm using a colorimetric plate reader (FLUOstar Omega Microplate Reader, BMG LABTECH, Cary, NC, USA). Using serial dilutions of an untransfected cell suspension at 2500, 5000, 10,000, 20,000, and 40,000 cells, a standard curve was generated by plotting the number of cells vs. A460. The mean cell counts were calculated per setup at each timepoint.