Cells were plated at a density of 1000 or 2000 cells per well in 96-well plates. The next day the growth medium was replaced with DCSS containing the hormone (E2) or other treatments (forskolin, 8-CPT cAMP, H-89 plus E2, SQ22 536 plus E2). Controls for E2 treatment were done on a separate plate, because we had previously determined that low amounts of volatilized estrogens can affect responses mediated via nongenomic signaling pathways ; control treatments were with the vehicle in which test compounds were solubilized (ethanol or dimethyl sulfoxide). In some experiments the growth medium was replenished every second day, and after 5 days the cells were fixed with 2% paraformaldehyde/0.1% glutaraldehyde in PBS. The number of the cells in each well was determined using the crystal violet (CV) assay , which we modified previously . Briefly, the cells were incubated in 0.1% CV for 30 min at room temperature, excess dye was removed by three brief rinses with ddH2O, the plates were air dried, and the dye was extracted with 10% acetic acid, which was then read in a plate reader (Wallac 1420; Perkin Elmer, Boston, MA, USA) at 590 nm.
The utility of the CV assay in measuring cell number was verified both in MCF-7 cells and in combination with immunodetection plate assays for GH3/B6 cells . We compared CV results with DNA content measurements and with cell number counts by hemocytometer and both assays correlated very well (data not shown). Additionally, we compared the CV method with the MTT assay (ATCC, Manassas, VA, USA), which is often used to determine cell number and viability. Different numbers of mERhigh and mERlow cells (1000–7000) were plated in 96-well plates, and fixed and treated with CV as described. Both sets of data were approximated with a single linear regression line (Fig. (Fig.1a).1a). The number of cells determined by CV versus MTT assays were linearly correlated (Fig. (Fig.1b1b). Full paper
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