Hi fellow researchers,
I am having trouble performing live cell imaging with lysotracker blue using LSM780.
Goal of my experiment is to track lysosome upon treatment, and I am using lysotracker blue (D-22). Although recommended concentration is ~75nM, I never got any good results with this. So last time I performed live cell, I used 250nM~1uM, 30min incubation at 37C. But it always gives me nucleus staining as if I stained with DAPI. Lysosome shows up better at this concentration, but there is always nucleus. For confocal, I use lysotracker setting, and laser is 2%, with pin hole slightly larger than 1 airy unit (~1.3um). Digital gain isn't high, and no subtraction since there is no noise other than nucleus. Does my setting look okay? How can I change in order to get rid of nucleus?
Now the nile red. Since I always have bleed through into green channel, I just add it directly into my MacTek. It goes in to the cells well, but it is always very fuzzy unless I have higher laser percentage (from 2 to 5%, some times 7%) and high digital gain. Then now there is too much noise, so i have to take away the background -500. For fixed cells, I never have to do this much manipulation, but it's always trouble for the nile red. Any tips on getting better live cell imaging for nile red staining?
Also for live cell imaging, is it better to use phenol red free media?
Thanks very much and have a great day.
What I do know is that you SHOULD use imaging media (no Phenol Red) for live cell fluorescence. That should substantially cut down on background fluorescence. I would try this first and see what happens.
I don't have experience with lysotracker, but what you are describing could be related to the filter cubes you are using, or to the cell type. Have you tried with other types of cells to see if you get less background? If you check your filter cube spectra, you'll see if there's something autofluorescence or bleed through issues related to those cubes. Alternately, if your 780 has the fancy tunable filter system, try using that to tune the bandpasses.
It's a silly idea, but maybe you could go stain with a live nuclear dye and use the difference between that and the lysotracker signal to isolate where the actual lysosomes are?
Dear Christina,
Thanks for your concerns. We are doing relatively short (30 min to 1hour) imaging and we are indeed using the same concentration of nile red as we use for staining fixed cells. We still get bleed through from fixed cells, but it is not as dramatic. Also, in fixed cells, if signal from green is strong, we can distinguish the positive cells better. Maybe in live cell we will decrease the concentration.
We have not yet figured out how to use lysotracker blue effectively. We did decrease the concentration and incubation hour, but still we get nucleus like staining. Manager in our imaging facility said it could be artifact from the blue channel and she recommends lysotracker of other colours.
Hi,
I have used 75 nM lysotracker blue for 30 min at 37 deg. In presence of phenol red containing DMEM, the fluorescence was quenched significantly. Replacing the media with PBS, just before imaging gives good results.
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