I am currently doing a NR cytotoxicity assay and I have encountered very low dye uptake even for my control cells. Evaluation under the microscope shows that the cells are still attached to the wells and the dye is seen in only some of the cells, however it is very light in color.
The protocol I used is as such:
1)Seeding of cells at 100000 cells/well in 96 well plates followed by treatment for 48 hours
2)A day prior to the assay, NR stock solution (40mg/ml) is used to prepare the NR media at 1:100 dilution with serum free media and left in the incubator overnight
3) On the day of the assay, the NR media is centrifuged and syringe filtered to remove percipitates.
4) Plate wells were emptied and the filtered NR media replaced. Plates were incubated for 3 hours
5) NR media removed and wells were filled with NR resob solution to solubilize the dye.
5) Absorbance read at 540nm
At the end of the assay, the maximum absorbance I got for the untreated control was aprox 0.2 and all 6 extracts i tested gave the exact same trend, even the positive controls.
I think that somehow there is a limiting factor that limits the availability of the NR for cellular uptake and i cant seem to find out what's the problem. I have tried reducing the dilution when preparing the NR media to 1:80 and incubation time to 4 hours but it didn't make any difference.Also, I did try fixing the cells with 1% formaldehyde in PBS buffer before extracting the dye but to no avail. Could the filtering be a problem? If it is, how else am I suppose to remove the precipitate as centrifuging alone does not totally pellet it?
I would be very grateful if someone could advice me.
Thank you in advance.
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Neutral Red Cytotoxicity Assay Trobleshooting
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