I'd like to test a cytotoxicity effect of a drug by MTT assay.
How density of A549 cells can I seed at 96 well-plate before treatment?
What compound used as a positive control for them?
Thank you!
Tran Dat
density 1 x 104 cells / ml (1 x 103 cells / well) for 96 hours incubation with compound
density 2 - 3 x104 cells / ml (2 - 3 x 103 cells / well) for 48 hours incubation with compound
positive control - cisplatin ( IC50 in my experience in A549 cells after 96 hours incubation forcisplatin was 3.4 uM)
You can seed 7000-8000 cells in 96 well plate for A549 because they grow very fastly.If you are doing starvation, 24h starvation is fine. Yes, trypan blue assay is more better option.
Be aware of pitfalls of the MTT assay. MTT reduction occurs throughout a cell and can be significantly affected by a number of factors, including metabolic and energy perturbations, changes in the activity of oxidoreductases, endo-/exocytosis and intracellular trafficking. Over/underestimation of cell viability by the MTT assay may be due to both adaptive metabolic and mitochondrial reprogramming of cells subjected to drug treatment-mediated stress and inhibitor off-target effects. I provided a comprehensive survey of factors that should be accounted in the MTT assay. To avoid result misinterpretation, supplementation of the tetrazolium salt-based assays with other non-metabolic assays is recommended (https://www.ncbi.nlm.nih.gov/pubmed/?term=Pitfalls+of+the+MTT+assay%3A+Direct+and+off-target+effects+of+inhibitors+can+result+in+over%2Funderestimation+of+cell+viability) (DOI:10.1016/j.gene.2015.08.009)
Hello
I agree with Hina Agraval
and see links here
Good Luck
http://www.ptfarm.pl/pub/File/Acta_Poloniae/2013/1/087.pdf
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2992145/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3022071/
I think 5000 cells/ well is fine and MTT measures general toxicity so I would say use both MTT and Trypan blue assay.
Good luck
density 1 x 104 cells / ml (1 x 103 cells / well) for 96 hours incubation with compound
density 2 - 3 x104 cells / ml (2 - 3 x 103 cells / well) for 48 hours incubation with compound
positive control - cisplatin ( IC50 in my experience in A549 cells after 96 hours incubation forcisplatin was 3.4 uM)
I totally agree with the comment "be aware of the pitfalls..." What is the point of determining the IC50 values by such assays? I have been publishing in this field since the early 1980's and recently decided to determine the impact of cancer cell enlargement on radiosensitivity and chemosensitivity as measured by multiwell plate assays (e.g., MTT, XTT, CellTiter-Blue, etc). When performed under optimal conditions, with solid tumor-derived cell lines, such assays do not measure loss of viability or cytotoxicity. In response to clinically relevant doses of genotoxic agents (including cisplatin), the emergence of highly enlarged cancer cells that remain viable and metabolically active (as determined by the single-cell MTT assay) complicates the interpretation of data obtained by multiwall plate assays. It is important to note that giant cancer cells that are developed following therapeutic exposures give rise to tumor repopulating progeny (e.g., through budding). To assume that IC50 values as measured by multiwall plate assays reflects loss of viability/cytotoxicity might be misleading, as we have recently shown to be the case for cisplatin-treated A549 cells. If you are interested to know the details of these observations, please search the PubMed for my name "Mirzayans R" and you will find a number of recent open access papers. Alternatively, please feel free to send me an email ([email protected]).
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