Hi, i need your insight on the following problem:

I have issues with my Neutral Red viability assay. I have a very high variation in my cell free controls when i do the Neutral Red staining. I work with an OECD guideline (249) , where i compare a cell free control with my highest test item concentration, with a cell free control which only contains medium.

My issue is, that i only have one well each and because of the high variation they sometimes have a variation of more than 20%, which makes my test invalid.

I prepared a whole plate, just with cell free controls. My variation differs from a value of 1% to up to >20%. I have no idea, why the values of my neutral red staining show such a huge variation. I thought it might be due to crystals, but i didnt see any, i filtered and i centrifuged, but nothing made a difference. My ph values were in the range of 7.1 and 7.2 so that shouldnt be an issue.

Do you have any idea, how i can get more comparable values for my cell free controls? I think it has to be some kind of issue of dye residue in the wells, because there are no cells, but i washed the wells two times and it didnt work.

I prepare my NR solution in PBS with a pH of 7.1 on the day of the test, i incubate the cells with the NR for 1h at 19°C, after that i add Fixation solution and extraction solution.

It would be great if someone has an idea! Thank you!