http://confocal-microscopy-list.275.s1.nabble.com/lysosomal-live-cell-markers-tp7581040p7581102.html
To best of my knowledge, all lysosomal dyes are weak bases that are initially neutral and are trapped in all sufficiently acidic (pH <6) vesicles by protonation to the conjugate acid. Unfortunately, my knowledge isn't complete as many of the probes you mention don't have published structures. I would not recommend using anything that does not have a published structure as you put yourself in the position of investigating one unknown using another. For weak base dyes like LysoTracker, the most obvious mechanism of toxicity is lysosomal alkalinization and the most direct countermeasure is to use the smallest possible staining concentration. In my experience, you can use LysoTracker Red at 10 nM on disseminated cell cultures. Higher concentrations are only really needed on tissue specimens. The staining is good for 1-2 hours in live cells. If you want to go 24 hours, GFP-LAMP is far superior to any dye-based method as long as your cells are amenable to transfection beforehand.
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> Hi all-
>
> I will be doing a series of experiments that involves tracking lysosomes by
> live-cell microscopy over 1-24 hours. I took a look at the listserv
> archives and hadn't seen much recently on the topic of live cell lysosomal
> markers. While LysoTracker has been a standard for a while, my
> understanding is that there are potential pitfalls, including purported
> photoswitching from red to green with LysoTracker is excited, potential
> toxicity, lack of specificity to lysosome (rather than just all acidic
> compartments), and low fluorescence levels.
>
> Compared to LysoTracker, how are some of the other non-FP based methods for
> live-cell lysosomal tracking? I'd be curious to hear thoughts on Enzo's
> Lyso-ID, MarkerGene's LysoLive lysosomal sulfatase kit, Abcam's CytoPainter
> lysosomal kit, AAT's LysoBrite (which they claim is much more photostable
> and less toxic than LysoTracker), Magic Red detection of cathepsin B
> activity, or preloading cells with fluorescently labeled dextrans?
>
> I'm specifically interested in:
> a) specificity of labeling
> b) toxicity
> c) photostability (resistance to bleaching and photoconversion)
> d) how long the label lasts if I were to do a prolonged time-course
> experiment (e.g. 24 hours)?
>
> Thanks so much for your insights in advance.
>
> -Jason Miller
>
> --
>
> Jason Miller, MD, PhD
>
> University of Michigan Kellogg Eye Center
>
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>
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