Celltiter 96tm aqueous non radioactive cell proliferation assay

Growth in vitro studies were conducted using the soluble tetrazolium salt MTS colorimetric assay (Cell Titer 96TM AQueous Non-Radioactive Cell Proliferation Assay, Promega, Madison, WI, USA). MTS couples fenazine methylsulfate (PMS) and is reduced to formazan. Both cell lines were seeded in 96-well microplates at a density of 5000 cells per well in RPMI medium supplemented with 5% FBS. After 24h, cells were deprived of FBS to induce quiescence (24 h previous to the proliferation assay) and were treated with increasing concentrations of P4 or ALLO (10 -11 -10 -5 M) by sextuplicate during 72 h. Later, the medium was replaced with 100 μl of fresh medium containing 20 μl MTS: PMS (20: 1), and after 2 h of incubation at 37°C, the production of formazan was evaluated by measuring the absorbance at 490 nm with an ELISA reader (Bio-Rad Laboratories, Inc. Oakland, CA, USA). Three independent experiment were made; and the mean absorbance value of the sextuplicate samples ± SEM were calculated for each experiment. Results were expressed as a percentage of stimulation in relation to control (cells without treatment). Moreover, alive and dead cells were analyzed through direct cell count with trypan blue [25] .