viability test: fixation AFTER propidium iodide staining? - (Oct/28/2009 )
Hi,
I have a killer toxin, which generates pores on susceptible yeast cells, so I want to make a viability test with PI. Now, I incubate the sensitive cells with the toxin for 12 and 24 hour, but the problem is that I can't afford to make the measurments with the flow cytometer at each sampling time. The question is that, after the sampling and staining, can the cells be stored at 4ºC simply, or I have to fix them with 70% ethanol or formaldehyde (which one do you prefer?). Will not the PI diffused from the dead cells at 4ºC overnight? I suppose it won't. What do you think?
Thanks in advance.
-maltosa-
maltosa on Oct 29 2009, 12:09 AM said:
Hi,
I have a killer toxin, which generates pores on susceptible yeast cells, so I want to make a viability test with PI. Now, I incubate the sensitive cells with the toxin for 12 and 24 hour, but the problem is that I can't afford to make the measurments with the flow cytometer at each sampling time. The question is that, after the sampling and staining, can the cells be stored at 4ºC simply, or I have to fix them with 70% ethanol or formaldehyde (which one do you prefer?). Will not the PI diffused from the dead cells at 4ºC overnight? I suppose it won't. What do you think?
Thanks in advance.
I have a killer toxin, which generates pores on susceptible yeast cells, so I want to make a viability test with PI. Now, I incubate the sensitive cells with the toxin for 12 and 24 hour, but the problem is that I can't afford to make the measurments with the flow cytometer at each sampling time. The question is that, after the sampling and staining, can the cells be stored at 4ºC simply, or I have to fix them with 70% ethanol or formaldehyde (which one do you prefer?). Will not the PI diffused from the dead cells at 4ºC overnight? I suppose it won't. What do you think?
Thanks in advance.
You definitely need to stain with PI just immediately before analysis. Otherwise, I think you can use Ethidium Bromide Monoazide that I suppose covalently binds to the DNA, and after this staining you can fix. Check on the web about the properties of this dye, because I'm not sure 100% ( I have never done this)
-canotto-