I am trying to reproduce the 3T3 NRU phototoxicity assay as described in the OECD guidelines, where the neutarl red assy is used for viability, and my absorption at 540 does not reach the expected/accepted levels. I have serious doubts as to wether the N of cells I am plating is enough, please help since it is very urgent that I obtain results. Any hints as how to improve the assay (variabilities of the protocol?). I am using normal ELISA (sterile) 96 flat-bottomed well platres, during the incubations the lid is on. I have used PBS for the washing, the neutral red has been prepared freshly every time (and filtered), I have tried leaving the cells to grow for 24 and 72 hours, the results were better at 24 than at 72 (possible decrease in the cells activity?).