Lysotracker Red

[Jean-Pierre CLAMME-2]

Hello,

I was wondering if some of you had experience with the LysoTracker Red DND-99 and maybe even on the Fluoview 1000.
I tried it out today and I was surprised by the weakness if the signal.  I had to use over 3% of my 559 Laser line (I generally use 0.3 to 0.5% for Cy3 labelled particles detected on the same channel).
It would be helpful if I could get some feed backs from your experience with this dye.

Thank you in advance,

JP


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Jean-Pierre CLAMME, PhD
Senior Scientist
Nitto Denko Technical
501 Via Del Monte
Oceanside, CA 92058
E-mail: [hidden email]
Phone: [phone]

[Cameron Nowell]

Hi JP,
 
I wouldn't be concerned about 3% power, in fact I would think that is quite low. I have some users using lyotracker red (and green) in whole zebrafish and they routinely are setting the 559 laser at around 10-20%. This is on an FV1000 system with the 20x NA0.95 dipping objective.
 
I have a feeling the lysotracker dyes are a bit weak, i would be interested in hearing other peoples experience as well. We have tried the blue/yellow in the past and have given up on it because while the signal is there, it is very weak and required high laser power and long scan times to get a decent image.
 
Cheers
 
 
Cam
 
 
 
Cameron J. Nowell
Microscpy Manager
Central Resource for Advanced Microscopy
Ludwig Insttue for Cancer Research
PO Box 2008
Royal Melbourne Hospital
Victoria, 3050
AUSTRALIA
 
Office: [phone]
Mobile: [phone]
Fax: [phone]
 
http://www.ludwig.edu.au/branch/research/platform/microscopy.htm
 

________________________________

From: Confocal Microscopy List on behalf of Jean-Pierre CLAMME
Sent: Sat 30/05/2009 10:58 AM
To: [hidden email]
Subject: Lysotracker Red



Hello,

I was wondering if some of you had experience with the LysoTracker Red DND-99 and maybe even on the Fluoview 1000.
I tried it out today and I was surprised by the weakness if the signal.  I had to use over 3% of my 559 Laser line (I generally use 0.3 to 0.5% for Cy3 labelled particles detected on the same channel).
It would be helpful if I could get some feed backs from your experience with this dye.

Thank you in advance,

JP


- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Jean-Pierre CLAMME, PhD
Senior Scientist
Nitto Denko Technical
501 Via Del Monte
Oceanside, CA 92058
E-mail: [hidden email]
Phone: [phone]

[Julian Smith III]

No experience with Lysotracker Red, but I normally run our 559 at 10%,
4ns dwell time on our FV-1000 spectral--either Cy3 or AlexaFluor 568.
Julian

Jean-Pierre CLAMME wrote:

>
> Hello,
>
> I was wondering if some of you had experience with the LysoTracker Red
> DND-99 and maybe even on the Fluoview 1000.
> I tried it out today and I was surprised by the weakness if the
> signal.  I had to use over 3% of my 559 Laser line (I generally use
> 0.3 to 0.5% for Cy3 labelled particles detected on the same channel).
> It would be helpful if I could get some feed backs from your
> experience with this dye.
>
> Thank you in advance,
>
> JP
>
>
> - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
> Jean-Pierre CLAMME, PhD
> Senior Scientist
> Nitto Denko Technical
> 501 Via Del Monte
> Oceanside, CA 92058
> E-mail: [hidden email]
> Phone: [phone]

--
Julian P.S. Smith III
Director, Winthrop Microscopy Facility
Dept. of Biology
Winthrop University
520 Cherry Rd.
Rock Hill, SC  29733

803-323-2111 x6427 (vox)
803-323-3448 (fax)
803-524-2347 (cell)

[Jean-Pierre CLAMME-2]

Hi Cameron,

Thanks for the infos. From what I tested on friday and from what you wrote, it's seams that for live cell imaging, Lysotracker won't be a good tool at all.

Thanks,

JP



> Hi JP,

> I wouldn't be concerned about 3% power, in fact I would think that
> is quite low. I have some users using lyotracker red (and green) in
> whole zebrafish and they routinely are setting the 559 laser at
> around 10-20%. This is on an FV1000 system with the 20x NA0.95
> dipping objective.

> I have a feeling the lysotracker dyes are a bit weak, i would be
> interested in hearing other peoples experience as well. We have
> tried the blue/yellow in the past and have given up on it because
> while the signal is there, it is very weak and required high laser
> power and long scan times to get a decent image.

> Cheers

>
> Cam

>
> Cameron J. Nowell
> Microscpy Manager
> Central Resource for Advanced Microscopy
> Ludwig Insttue for Cancer Research
> PO Box 2008
> Royal Melbourne Hospital
> Victoria, 3050
> AUSTRALIA

> Office: [phone]
> Mobile: [phone]
> Fax: [phone]

> http://www.ludwig.edu.au/branch/research/platform/microscopy.htm

>
> ________________________________

> From: Confocal Microscopy List on behalf of Jean-Pierre CLAMME
> Sent: Sat 30/05/2009 10:58 AM
> To: [hidden email]
> Subject: Lysotracker Red

>
> Hello,

> I was wondering if some of you had experience with the LysoTracker
> Red DND-99 and maybe even on the Fluoview 1000.
> I tried it out today and I was surprised by the weakness if the
> signal.  I had to use over 3% of my 559 Laser line (I generally use
> 0.3 to 0.5% for Cy3 labelled particles detected on the same channel).
> It would be helpful if I could get some feed backs from your
> experience with this dye.

> Thank you in advance,

> JP

>
> - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
> Jean-Pierre CLAMME, PhD
> Senior Scientist
> Nitto Denko Technical
> 501 Via Del Monte
> Oceanside, CA 92058
> E-mail: [hidden email]
> Phone: [phone]

[George McNamara]

Hi Jean-Pierre,

How acidic are the organelles that you are imaging? LTR accumulates in acidic compartments. I was involved in an experiment where we used LTR and the culture medium was moderately bright orange by eye (i.e. extracellular LTR is a nice "negative stain" for live cells), the cytoplasm was dark, and lysosomes, etc, were much brighter than the culture medium. Bottom line: leave LTR in the culture medium and adjust your expectations.

good imaging,


George

At 08:58 PM 5/29/2009, you wrote:

Hello,

I was wondering if some of you had experience with the LysoTracker Red DND-99 and maybe even on the Fluoview 1000.
I tried it out today and I was surprised by the weakness if the signal.  I had to use over 3% of my 559 Laser line (I generally use 0.3 to 0.5% for Cy3 labelled particles detected on the same channel).
It would be helpful if I could get some feed backs from your experience with this dye.

Thank you in advance,

JP


- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Jean-Pierre CLAMME, PhD
Senior Scientist
Nitto Denko Technical
501 Via Del Monte
Oceanside, CA 92058
E-mail: [hidden email]
Phone: [phone]