I need a non-cytotoxic / non destructive way (i.e. a dye or measuring something within the media) to determine # of live epithelial cells in sheet grown on a transwell filter. Ideas?
My exposure regimen requires 12well plates so the plate won't fit in a fluorimeter.
I'm concerned that a dye like crystal violet could be incorporated but the cell not detach (due to sheet formation) resulting in artificially hi counts. Also have read that crystal violet can damage cells
Could look at 'change from untreated' as a measure (rather than an actual count of cells) but the cells must grow for 24hr after treatment before analysis meaning the marker molecule would be hanging out in the media for 24hr so I am not certain that something like LDH assay would be possible/useful.
This is not a migration assay-- just one epithelial cell type.
Advice? Thanks in advance!
Hi Karen,have you tried to stain transwells with Crystal violet?if the cell are alive, the should be bright and with, if not blue..
Giacomo
I haven't tried CV yet bc of my concerns. I have heard CV causes damage (need to use only as an endpoint assay), and concerned that dead cells may stay in the epithelial sheet due to junctional complexes.
CV binds 'proteins' does it only enter live cells? Also the protocols I have seen all include methanol fixation steps-- can the cells be stained and counted while alive?
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