I've been working with some adherent cells (cancer cells) and trying to use an MTT assay to evaluate % survival 3 days after transfection with lipofectamine and miRNA mimics. I'm plating cells in a 6 well plate (20-25e103 per well) and waiting 24 hrs. then transfecting cells for 4hrs. After 4hrs. I'm removing the medium (Lipofectamine, Opti-MEM, mimics) from the cells and replacing with complete growth medium for 72 hrs. At 72 hrs. removing the complete growth medium and adding MTT to the cells incubating 1-3 hrs. at 37 degrees in CO2 incubator. When the cells turn blue I remove the MTT and add DMSO to the cells, mix the cells via pipette. Transfer the cells to a 96 well plate and read the absorbency at 570nm on a plate reader. Is there something critical that I am missing in my MTT protocol? Thank you for any input.
Hi
why do you transfer the cells to a 96 well plate and read the absorbency instead of reading the absorbency in their 6 well plate?
Thank you Ana. I am working with cancer cells and as I research various methods to running the assay, I'm seeing that some protocols leave the MTT in the wells and add DMSO and read at various wavelengths. I wanted to see what types of protocols other labs are using and generating successful results. I will look at the kit that you are using. Thank you for the information.
I would recommend Resazurin assay instead of MTT. Similar principles, better workflow, less expensive. It is available in kit format (http://www.cellsignal.com/products/cellular-assay-kits/11884 and others) and you can get it in powder from major vendors like Sigma and alike.
Here is areference for resazurin: http://en.wikipedia.org/wiki/Resazurin
You can also try using MTT solubilization solution instead of DMSO. It works fine with me.
Here are the compositions:
To make 50ml:
1) 5mL Triton-X 100
2) 45mL Isopropanol
3) 1 Drop HCl 37% (12M)
Hi
why do you transfer the cells to a 96 well plate and read the absorbency instead of reading the absorbency in their 6 well plate?
Hi Mahdi,
Yes, I transfer the cells from a 6 well plate to a 96 well plate to read them. I was able to get the assay to work last week. It turns out that I was not diluting the samples with enough DMSO. I'm giving it another try this week. Thanks everyone.
Hi Mahdi,
I transfer to a 96 well plate because the plate reader has a template set up for a 96 well format and not a 6 well.
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