lysosomal live cell markers

[Jason Miller]

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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

E-mail: *[hidden email] *


The first thing which I can record concerning myself is, that I was born.
These are wonderful words. This life, to which neither time nor eternity
can bring diminution - this everlasting living soul, began. My mind loses
itself in these depths.  -- Groucho Marx

[Cliff Luke]

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I have always used Dextrans (10,000 MW) or BSA (TMR or Alexa Fluor647
Labeled) on both cells and C. elegans for the following reasons:

1. they are non-toxic
2. Last for days - (4-5 days)
3. very photostable

Of course the caveat is that they are taken into the lysosomes via the
endocytic pathway and require a chase (>4 hours) after labeling to ensure
endosomes are not labelled as well. I get great colocalisation with
cathepsin L and other lysosomal enzymes.

Hope this helps,

Cliff

[hidden email]

[John Naleway]

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Dear Jason,

From our experience, the MarkerGeneT LysoLiveT Lysosomal Metabolic Health
Assay Kit http://www.markergene.com/M1910.php , Product M1910 has the best
properties for what seems like your application.

It's been validated versus LAMP1 and LAMP2 staining, is extremely bright and
relatively photostable and exhibits significantly less toxicity than
LysoTracker.  Because it's an enzyme substrate, it will accumulate in the
cells, and some optimization might be needed since it utilizes a kinetic
assay format.

Cheers,

John


----- Original Message -----
From: "Jason Miller" <[hidden email]>
To: <[hidden email]>
Sent: Friday, September 27, 2013 10:26 AM
Subject: lysosomal live cell markers

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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
>
> E-mail: *[hidden email] *
>
>
> The first thing which I can record concerning myself is, that I was born.
> These are wonderful words. This life, to which neither time nor eternity
> can bring diminution - this everlasting living soul, began. My mind loses
> itself in these depths.  -- Groucho Marx
>

[Kilgore, Jason-2]

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** Vendor Reply **

Pretty much any lysosomal marker other than FPs will have some effect on the cell which will lead to toxicity, given enough time.  With LysoTrackers, it could be a disruption of lysosomal pH.  Enzyme substrates, too, would surely have an effect (due to binding of enzymes that should be used elsewhere).

That being said, for most standard imaging periods (within an hour or so of the incubation period), we haven't seen any significant cell toxicity for most cell types, but there may be some sensitive cell types out there.  Of course, the higher the concentration and the longer the incubation period, the more likely you are to see toxicity.  For the LysoTrackers, a label time and concentration of only 1-5 minutes at 100 nM, in HBSS, is usually sufficient.  In any case, I haven't seen any publications demonstrating toxicity with LysoTrackers.  

Regarding specificity, LysoTrackers have been shown to be highly selective to lysosomes in a number of tests, including colocalization with FPs.  Email me offline if you would like further information on this.

LysoTrackers are not the most stable of fluorescent dyes, but they haven't been problematic for most imaging applications.  In my experience, LysoTracker Red is the most photostable (though I haven't tested the deep red version for photostability, myself).  

A previous commenter (who is president and CEO of Marker Gene Technologies) had posted a reply about photoswitching of LysoTrackers, but this only occurs with high-intensity illumination, such as useful for SRM modalities like STORM (example paper: http://www.ncbi.nlm.nih.gov/pubmed/22891300).

With dextrans, photostability depends on the dyes.  The Alexa Fluor versions are certainly king, in most cases.  And I'm not aware of any toxicity issues with using dextrans.  For long-term imaging, outside of FPs, dextrans would therefore be an excellent choice.

For more discussion on lysosomal labeling reagents (as well as other vesicular structures and BacMam-based FP detection), you can see the following recent reference that I co-authored with Nick Dolman and Michael Davidson:
http://www.ncbi.nlm.nih.gov/pubmed/23835803

Cheers,

Jason  (with feedback from colleague and fellow confocal listserve member Nick Dolman)

Jason A. Kilgore
Technical Application Scientist
Molecular Probes Labeling and Detection Technologies
Cells Systems Division
 
T 1 800 955 6288 then option 4, then option 6,  or  541 335 0353 * F 541 335 0238
29851 Willow Creek Rd * Eugene * OR * 97402-9132 * United States


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jason Miller
Sent: Friday, September 27, 2013 10:27 AM
To: [hidden email]
Subject: lysosomal live cell markers

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

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

E-mail: *[hidden email] *


The first thing which I can record concerning myself is, that I was born.
These are wonderful words. This life, to which neither time nor eternity
can bring diminution - this everlasting living soul, began. My mind loses
itself in these depths.  -- Groucho Marx

[Iain Johnson]

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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.

Iain

Sent from my iPhone

On Sep 27, 2013, at 10:26 AM, Jason Miller <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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
>
> E-mail: *[hidden email] *
>
>
> The first thing which I can record concerning myself is, that I was born.
> These are wonderful words. This life, to which neither time nor eternity
> can bring diminution - this everlasting living soul, began. My mind loses
> itself in these depths.  -- Groucho Marx