Hi! I would like to adapt the FDA/PI live/dead method to assess the viability of airways cells in PET membranes. Has anyone adopted this method on transwell cultures and can confirm it works. Should I stain cells from the apical compartment only or also the basolateral compartment.
Many thanks
Hi James I've grown cells on PET cells quite a bit (I use them for laser dissection).I generally grow them on the slides in the incubator (all but neuronal cells will stick down more or less - for neuronal you need to coat the membrane with Laminin).
I the incubate the cells in 4-8 uM Calcein AM dye (from Thermo - it is in their "live-dead" viability kit https://www.thermofisher.com/uk/en/home/brands/molecular-probes/key-molecular-probes-products/live-dead-viability-brand-page.html but can also be purchased separately)
This will lead to an accumulation of fluorescence in the live cells (I use Calcein green but other moieties exist). I also use this channel (488 nm) to focus on the cells for cutting.
Personally I dislike PI as it also stains RNA so I would use ET BR (which is what they provide in the above kit).
You could stain the cells differentially (i.e. apical green and basolateral blue) to distinguish the origin of the cells.
Calcein fluorescence however is sensitive to light and I am not sure of it's persistence in non-fixed cells so may not be suitable for long-term migration assays (so FDA perhaps better - however I would still use EtBr vs PI).
Hope this helps
Gary
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