I am trying to reproduce the 3T3 NRU phototoxicity assay as described in the OECD guidelines 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, plaes help since it is very urgent that I obtain results. Any hinys 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?).