The idea of fixing the cells with 4% Paraformaldehyde/ ethanol is to make sure the cells do not wash off and are still present during the whole process .
So if I wanted to do this experiment the steps I would follow would be to
1. Remove media in which cells are initially growing
2. Wash gently with PBS without disrupting cells too much
3. Fix with 4% PFA for 10mins at room temperature
4. Incubate with mix of Calcien-AM and EtBr in PBS/serm free media for 30mins @ 37degrees
Now my doubts are should i skip step 3 and just go directly to step 4 or what other steps do I need to include so my staining works perfectly.
both your dies will penetrate the cells either way, so they won't need any form of fixing or permeabilization.
so you probably can skip step 2 also then, reducing your chances of losing cells...
(or wash with medium without FBS that has been preheated to 37 degrees so your cells stay "happy" if you're worried the low amount of remaining FBS would have an influence)
I do not think Calcein-AM will work on fixed cells. The flouresecene is generated by the esterase enzymes in live cells. So you should not be fixing your cells
I concur the above two comments. At least calcein is a vital stain which do not need fixation. Attached cells usually do better with gel and will not be easily washed off unless you intentionally wash them off forcefully.
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