dear everyone,
How to analyze mammosphere cell death using the PI staining. I am planning to use a FACS analysis but I am affraid to get false results after forcing cells constituting mammospheres to separate to single cells.
Thank you.
Dear Hamadi, you can use flow cytometry for analysing mammospher viability by individual cell analysis or by whole mammosphere analysis. First method, as you mention, imply mammosphere separation, but you can try a very light method using EDTA and agitation, that produces a more or less individual suspension, and then stain and run in a flow cytometer. The other method, is more tricky because you need to know the size of your mammospheres and the size of you flow chamber. Usually if your spheres are lower than 25% of flow chamber tip size, you can stain directly your spheres suspension and analyze them, even you can define cell number per sphere and quantify using controls of permeabilized pheres. If not, then you have to use the first method. Keep in mind that PI unbind from nucleic acids if you retire the PI from cell solution. Hope it helps, best regards, Alberto
Hello Hamadi,
I will suggest you to use standadrd trypsin-EDTA to make single cell suspension. Trypsin at a mild dose don't induce cell death as tht only do a reversible damage to integrins only and it doesn't result into cell death. Anyway we always do trypsinisation of our routine cell cultures and that don't induce any cell death into cells. So it is a misconception that trypsin will induce cell death. Only a harsh handling will do that.
Second, if you want to do microscopy, I will suggest you to club Acridine orange (vital live stain) with PI and then you can get a true picture of live and dead cells.
Hope it helps !
Hi,
No need to worry about the trypsin unless it is too much! Go for that, make single cell suspension and then do flow cytometry. Your problem will probably be getting desired number of cancer stem cell (or lets say cancer stem cell-enriched population) as it is not easy to get enough number of them for flow cytometry! So, we always do as said above. No worries.
There is a milder way for spheroids dissociation. Use Accutase cell detachment solution. We use it for dissociation of neurospheres generated from human glioma stem cells with very good results.
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