Is MTT appropriae for...

7 posts / 0 new
Last post
nani
nani's picture
Is MTT appropriae for...

I'm trying to illucidate the role of downstream signals of EphB receptor tyrosine kinases in cancer survival. In this regard I would like to know that, within short term exposures of receptor with its cognate ligand ephrin  ( e.g 15min to 2hrs ), would performing MTT assay be sort of appropriate one to determine the effects of a receptor activation and its downstream signals on  overall viability of cancer cells and see if the signals toxic to the cells or not? if so,when is the best time to start MTT assay after the desired time exposure of ligand and receptor has passed?
I'll appreciate it if any one can help me find my answer.
Thanks
 

samm
samm's picture
MTT will give you an idea of

MTT will give you an idea of the redox potential, and therefore the viability/mitochondrial health of your cells. Do note that its not the most sensitive assay out there, but especially in the XTT versions (no solubilization), it is very convenient.
As for your assay itself, you'll probably have to do a kinetic assay for yourself - different exposures, followed by atleast 2 h of XTT., with multiple readings at 4, 6h.
An alternative would be Alamar Blue, which will allow you to track each condition (eg 15 min exposure v/s 2 h, track 1h -24 h) for extended periods.

nani
nani's picture
Thanks for your great offer,

Thanks for your great offer, I studied about Alamar blue & I think I'm going to use it. As I understood Alamar blue is the commercial form of Resazurin sodium salt and they seem to have similar properties but I'm not sure which one is more reliable and need your kind help.
My other question is that in order to perform a kinetic assay using Alamar blue, is it possible to have multiple redings (after the desired periods of  receptor  stimulation has passed) at different time points with the same plate or various plates should be prepared and read at different times.
Just one more question: from your experience, how long will it take for cells to show their proliferation status with Alamar blue (i.e. how much time should pass after each time exposur to affect metabolism of the cell and thus reduction of Alamar blue)?
So much thanks for your generous help

samm
samm's picture
Hi Farnazb! The best thing

Hi Farnazb! The best thing about Alamar blue is using the same plate for several hours, with a gap between readings (equilibriation purposes - the data sheet will give you more info).  Sadly, I cannot give you a definitive answer based on cell type and assay, but if you set up 2 or 3 plates, you can alternate between them to take assays every 60/40 minutes if that helps - for upto past confluency (for untreated control cells). You'll get lots of data points that way - in my opinion, thats a very good thing!

samm
samm's picture
I think the Alamar Blue

I think the Alamar Blue format is supposed to be a more stabilized form of the resazurin salts that are a part of some microbiological culture media. Thats what enables readings even after you take it out of a CO2 incubator. All the best for your assays!
-sam

nani
nani's picture
Hi Samm, Thanks for your

Hi Samm, Thanks for your perfect ideas, Thus I'm going to get started that way.
best wishes from farnaz

nani
nani's picture
Hi samm,

Hi samm,
Thanks for sharing your valuable experinences to help others. This is really encouraging when one is frustrated.
I have a question about the interesting dye, Alamar blue, that I noticed just today in selecting an appropriate culture medium for MDA-MB-231: My choice is DMEM-High glucose + glutamin & pyrovate. Does existing phenol red in this culture interfere with Alamar blue colorimetric absorbtion and maybe other problems or they are Ok with each other?
 I think have to ask you more questions about Alamar blue assay in near future thus formerly sorry for the bother.