Hi
Does anyone knows a colorimetric (or relatively easy) assay to specifically determine growth inhibition?
I´ve seen that some persons use MTS, crsystal violet, tripan blue, etc. But this assays are used to evaluate cell death, metabolic activity, etc., not growth inhibition/proliferation directly.
I was considering the idea of using propidium iodide coupled with flow cytometry, but i want to know if there is an easier-less expensive option.
Any help will be highly appreciated.
Hey Mauricio,
as far as i know Propidium Iodid is used to mark dead cells via it's mechanism of intercalating into the DNA of dead cells.
Most colorimetric assays I know assess indeed the viability/metabolic activity which can be a indirect measure for Proliferation (more or less).
To directly measure the Proliferation you could eperiment with 3H-Thymidine which is incooperated during Proliferation. You can measure the Radio activity (week beta Radiation) at the end of your Experiment. Another Option could be CFSE labeling the cells and analyze them via flow cytometry.
You could also use Proliferation markers like Kir-67 or others (cell cycle markers for example) to determine the Proliferation via FACS.
hope this helps a little
best regards
Robert
I agree with robert. And in my experience CFSE alone is the best dye for monitoring cell proliferation. You just have to monitor the dye dilution in FITC channel. More dilution means proliferation and less or no dilution means less proliferation or growth. For more details, you can refer to the following.
http://onlinelibrary.wiley.com/doi/10.1002/cyto.a.22403/abstract
https://www.nature.com/nprot/journal/v2/n9/full/nprot.2007.296.html
Hi Mauricio,
try CyQUANT Cell Proliferation Assay - it looks exactly what you need.
It's some kind of non-radioactive replacement to 3H-Thymidine assay.
https://www.thermofisher.com/us/en/home/brands/molecular-probes/key-molecular-probes-products/cyquant-cell-proliferation-assays/cyquant-direct-cell-proliferation-assay.html
or
https://www.thermofisher.com/se/en/home/brands/molecular-probes/key-molecular-probes-products/cyquant-cell-proliferation-assays.html
Not expensive, reliable and simple.
Good luck!
Elvira
Hi Elvira.
I was wondering which would be the effect of quiescent cells on false positive signal in this assay, since these cells are not dead (They will enter G0 from what i´ve read).
I guess this assay will be very similar to the MTS assay in regard to the false positive signal generated by quiescent cells. In cyquant, quiescent cells will be labeled and will give signal (unless these cells´membrane permeability is compromised somehow). In MTS quiescent cells will still have metabolic activity (thus giving signal), but it will not be as strong as dividing cells.
I guess it all comes down to the amount of variables affecting growth or metabolism in general. If it´s not very expensive i guess i´ll try the Cyquant.
Thank you all for the information provided
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