I'm trying to optimize the NRU assay on 3T3 cells as low OD540 reading (about 0.1-0.2) were obtained from previously done experiments. I prepared 50ug/ml stock and incubated it at 37C for 1.5 hr, added to cells and incubate for 3 hr.
- I took the neutral red solution that's incubated at 37C from waterbath into the hood prior to washing of cells with PBS. Therefore, I'm thinking if the solution might get a little bit cool down (less than 37C) while I was washing the cells with PBS and that caused precipitations.
Yes, they look like neutral red crystals, try heating stock to 40-42°C for some time instead of 37°C, followed by 0.45uM filtration. It may help.
Thank you Vikas Taank
I'm in the midst of trouble shooting this assay with different variables, (e.g. different incubation time for stock solution, centrifugation, as well as filtration). I will also try your valuable suggestions, hope the nr crystals won't appear anymore!
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