Hello, researches,
So I am trying to do a cytotoxicity assay with virus-infected and inhibitor incubated cells. I would like to do quantification alongside MTT. I will have to kill the virus with PFA first, which will also permeabilize the live cells and propidium iodide will be all over the place then. Is there any fixation method that will not damage the live cell membrane?
P.S.I would like to do FACS and also microscopy.
Thanks in advance.
I would suggest going another way, you can count the population of the live cells versus the total cell number. In this case, you can use Calcein AM that is fixable. So first you incubate the live cells with Calcain for 15 minutes where the cells become fluorescent. Then fix the cells with PFA 1% for 4 minutes fixation will not affect Calcein fluorescence. Then you can run your flow to FITC 488 channel you will get the number of total events i.e. 10000 and a population of 488 positive events i.e. 5300 this will give you the viability of your culture of 53% or you can subtract this value from 100 to get death.
I would suggest to stain your cells before to fix them; we are using this procedure for PBMCs to add the live/dead staining...
Anton Lennikov Thank you so much for this information. Using Calcein sounds promising in my case. We have already discussed it in the lab today and looking forward to having it. Thanks again. Will try and input about the outcome.
Alessandro Torelli Yes now we are thinking of this. I think I will try both PI and Calcein. Hope it works.
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