Hi friendly people!
I've been doing live imaging of MDCK epithelial migration and I've noticed extreme variability in Hoechst staining. I use the NucBlue formulation where you just add 4 'drops' of staining solution to media and incubate the cells for 30 minutes at 37 degC before changing media and imaging. For several weeks, this has worked remarkably well and allowed live nuclear imaging during overnight experiments with low exposures/intensity and no apparent bleaching. However, I recently thawed a new batch of cells that look great in every way except that the Hoechst stain vanished over the course of an overnight run. As far as I know, I haven't changed my protocol, so I'm wondering if there's something important about Hoechst staining that I'm not aware of.
Anyone have a similar experience?
Thanks!
Daniel
Only thing I could think is that if you give just a pulse of Hoechst it should be diluted over time as the cell synthetize new DNA and divide. Have you noticed any difference in proliferation rate of your old batch of cells compared to the new one? I think that keeping Hoechst during live imaging in the medium would solve the problem, the issue is that you should find the optimal concentration and find a balance between DNA staining efficiency and cell death.
Hope it helps
Good luck
Hoechst has very low cytotoxicity. I would keep it in the medium during the course of your imaging and see if it helps. I've used NucBlue Live extensively and it is quite photostable so long as you are keeping your excitation intensity low. Hoechst emits very brightly, so you should be able to use very, very little light to get your signal.
Hi,
I am wondering if the new batch of cells are relatively more drug resistant due to somehow higher p-glycoprotein (or other MDR proteins?) expression that may pump the Hoechst out of the cells? This could actually be possible!...
Problem solved, sort of!
Thanks for your advice, but it turned out to be one of those ridiculous problems that could not have been predicted: our microscope software controller decided it would be a brilliant idea to gradually dim the LED excitation source over the course of the run and this produced the effect of Hoechst vanishing. We've seen it happen with other experiments, so we can confirm it was a software problem!
Re: leaving NucBlue in the media, I used to do that and it definitely stayed bright, but we had anecdotal evidence that cells didn't like it. However, our camera is more sensitive now, so we might be able to get away with that using lower excitations. We'll give it a shot, thanks!
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