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.
Please how long does the staining stay on cells, can you still image after 24hrs and the stained cells will still retain their stain?
Dear Hakeem,
You still need the fixation with 4% PFA (step 3) and the incubation for about 20 minutes should be after the staining by Calcien-AM and EtBr in PBS. The last step before imaging is to wash the cells with fresh media.
Hoping this answer clarified your point.
Rafik
Hi Rafik,
Thank you so much. Yes you did clarify my question. I am deeply grateful for your help.
Hakeem
Dear Rafik,
Please how long does the staining stay on cells, can you still image after 24hrs and the stained cells will still retain their stain? Thank you in advance.
Hakeem
Dear Hakeem,
I am not sure about what do you want to do with this protocol. Calcein AM is a cell-permeable dye mostly used to stain viable cells. Therefore, you can not fix your cells before staining them with calcelin AM. Since calcein AM need a viable cytosolic sterase for becoming fluorescent, I don't think you are able to see green if you previously fix your sample.
In my opinion, if you want to see viable cells: steps 1, 2 and 4 (without 3). Nevertheless, there are other options to distinguish alive from dead cells besides calcein AM (just in case you have troubles with this protocol). If you want to see all cells from your experiment, used the entire protocol but only with EtBr, Hoechst, or PI in step 4, to visualize cell nuclei from all (alive and dead) cells.
Regards,
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