I am trying to stain #ovarian cancer cells grown on gel with ethidium bromide and calcien. I am not too sure whether to fix my cells with 4% PFA or ethanol before staining. Any suggestions will be greatly appreciated
Thank you so much @Sebastian Schmitt for your input. This is really helpful.
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.
Thank you for your earlier response @Sebastian.
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.
Thank you so much Sebastian.
Please how long does the staining stay on cells, can you still image after 24hrs and the stained cells will still retain their stain even if placed in the medium? Thank you in advance.
Thank you so much Sebastian, your response were really helpful. I will take not of this and see how it goes.
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