I'm trying to do a live/dead cell assay (grown on 96 well plates) and I have problems with my cells adhering to the plate. I'm trying to establish a method that will allow addition of live/dead detection reagents, and then immediate fixation without washing out the live/dead reagents.
I need to fix the cells because this is for high throughput screening and I can't image quickly enough for accurate comparison.
Any thoughts?
Thanks Artur, but the problem is that my imaging machine is slow and I need to be able to image many plates of cells exposed to an external stressor for an equal amount of time.
So fixing the cells at end-stage is the best option to allow me to image them over a longer time period later on.
Perhaps you should consider fixing the adherence issue instead? That way you can get rid of your live/dead cell dyes before fixing. How about pre-coating the wells with poly-D-lysine or something similar? That always helps me when I have a tricky cell line that doesn't want to stick to the plate. Alternatively, you can process your plates in batches so they will have had the dye in the same amount of time when you do your imaging?
you can use 'CellTracker™ and CellTrace™ Reactive Probes' from Fisher. Live cells will be fluorescent and this compound is stable after fixation...
good luck!
Christina, I do coat with PDL, and I agree fixing the adherence would be better but is very tricky! I have tried...
Valentine, what are the catalog numbers for those reagents and have you personally found them to work well?
I used in the past Calcein AM green, that integrate only live cells, but this is not super stable after fixation (I still see them after PFA fixation). I know that other person in my lab used cell tracker and it works in their hand, but the purpose was totally different..
I think if you do a DAPI/cell tracker and compare dapi+/celltraker+ versus dapi+/celltraker-, it should work.
CellTracker™ Red CMTPX Dye Catalog number: C34552
you can write to their commercial to ask for a sample?
good luck!
https://www.thermofisher.com/us/en/home/references/newsletters-and-journals/bioprobes-journal-of-cell-biology-applications/bioprobes-issues-2011/bioprobes-66-october-2011/cell-tracking-dyes-qdots-fluorescent-proteins.html
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