I am currently using MTT assay to determine the optimal concentration of my mushroom extract and L-buthionine sulfoximine (BSO) on human fibroblast. I treated the cells with a range of concentrations of my extract or BSO for 48 hours. Following the treatment, I added 0.5 mg/mL of MTT to the cells and incubated it for 4 hours. I have noticed that some of the wells exhibit an abnormally strong purple hue compared to the other wells prior to the addition of DMSO and reading of absorbance. When observed under the microscope, the cells seem to be dead (detached and loss of spindle shape), however, a purple hue can still be observed.
I wish to know what may be the cause of this so that I may avoid it in the future. Thank you.
Hi Michael Phang
I think the issue here is chemical interference with your MTT assay. Mushroom extract sounds like there is a lot of different compounds in there and if any (e.g. - cyanide) uncouple oxidative phosphorylation from ATP production, this would affect the results of an MTT assay.
In addition, BSO reduces glutathione - this means that BSO can non-enzymatically reduce MTT to formazan, giving you a false positive.
With this in mind, you can also check cell viability via a trypan blue assay to confirm if your unattached fibroblasts are definitely dead but I would look at performing a different assay to test for optimal concentrations.
Hope this helps!
Michael,
Purple or violet coloration is the formation of grains smeared with cells.
On the contrary, pharmacan form viable cells that are capable of metabolizing MTT.
This means that your mushroom extracts stimulate fibroblast survival or metabolism. After the formation of pharmacan in fibroblasts, you need to add DMSO to these wells and thoroughly peptide until the pharmacan is completely dissolved.
Sincerely,
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