Lysotracker question

[Cecile Chalouni]

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

Hi everybody,

We are commonly using lysotracker to visualize acidic compartments which includes different types of endocytic compartments in addition to the lysosomes. I wonder if anybody knows what types of compartments are labeled with the lysotracker or/and at what pH it starts emitting?

Thank you in advance for your feedback!

Best regards,

Cecile


Cécile Chalouni, Ph. D
Genentech, Inc.
1 DNA Way, Mail Stop 72b
So. San Francisco, California 94080

p: 650-467-6772
c: 650-892-4763
e: [hidden email]

[sunandod]

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

Hi Cecile
   Sorry, I would not be of help but would like to get feedback on the
same question. In case you receive any postings, would you kindly
forward to my address?
We are also using Lysotracker RED DND to probe acidic compartments in
the enteric amoeba, Entamoeba histolytica and the amount of Lysotracker
we are using is quite different from what is generally being used for
mammalian cells.

Thanks in advance
Sunando
On 10/26/2013 2:42 AM, Cecile Chalouni wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi everybody,
>
> We are commonly using lysotracker to visualize acidic compartments which includes different types of endocytic compartments in addition to the lysosomes. I wonder if anybody knows what types of compartments are labeled with the lysotracker or/and at what pH it starts emitting?
>
> Thank you in advance for your feedback!
>
> Best regards,
>
> Cecile
>
>
> Cécile Chalouni, Ph. D
> Genentech, Inc.
> 1 DNA Way, Mail Stop 72b
> So. San Francisco, California 94080
>
> p: 650-467-6772
> c: 650-892-4763
> e: [hidden email]

[Iain Johnson]

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

Cecile:

LysoTracker Red has a pKa of 7.5 (see Duvvuri et al (2004) free online at
http://www.jbc.org/content/279/31/32367.long).  This means that the dye
will stain any vesicle with an internal pH of less than about 6.5 (at pH
6.5, >90% of the dye will be in the acidotropic protonated form).  Thus, in
addition to lysosomes (which typically have an internal pH of about 5),
other types of less acidic vesicle are amenable to staining (a fancy way of
saying the dye is not lysosome-specific).  I think the other LysoTrackers
are basically the same as they all have the same titratable substituent
(N,N dimethylaminopropane).  LysoTracker fluorescence is not pH sensitive
in the range 4-9. So a wash step is always necessary.  If you want a dye
that is pH sensitive (i.e. will not fluoresce significantly except in
acidic environments) then LysoSensor Green DND189 (Molecular Probes) is
probably your best bet (pKa = 5.2 therefore >90% nonfluorescent form at pH
>6.2).

Iain

Iain Johnson Consulting
Eugene, OR



On Fri, Oct 25, 2013 at 2:12 PM, Cecile Chalouni
<[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi everybody,
>
> We are commonly using lysotracker to visualize acidic compartments which
> includes different types of endocytic compartments in addition to the
> lysosomes. I wonder if anybody knows what types of compartments are labeled
> with the lysotracker or/and at what pH it starts emitting?
>
> Thank you in advance for your feedback!
>
> Best regards,
>
> Cecile
>
>
> Cécile Chalouni, Ph. D
> Genentech, Inc.
> 1 DNA Way, Mail Stop 72b
> So. San Francisco, California 94080
>
> p: 650-467-6772
> c: 650-892-4763
> e: [hidden email]
>

[Cecile Chalouni]

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

Thanks a lot Iain!

This is very helpful!

Best,

Cecile
On Oct 25, 2013, at 10:48 PM, Iain Johnson wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Cecile:
>
> LysoTracker Red has a pKa of 7.5 (see Duvvuri et al (2004) free online at
> http://www.jbc.org/content/279/31/32367.long).  This means that the dye
> will stain any vesicle with an internal pH of less than about 6.5 (at pH
> 6.5, >90% of the dye will be in the acidotropic protonated form).  Thus, in
> addition to lysosomes (which typically have an internal pH of about 5),
> other types of less acidic vesicle are amenable to staining (a fancy way of
> saying the dye is not lysosome-specific).  I think the other LysoTrackers
> are basically the same as they all have the same titratable substituent
> (N,N dimethylaminopropane).  LysoTracker fluorescence is not pH sensitive
> in the range 4-9. So a wash step is always necessary.  If you want a dye
> that is pH sensitive (i.e. will not fluoresce significantly except in
> acidic environments) then LysoSensor Green DND189 (Molecular Probes) is
> probably your best bet (pKa = 5.2 therefore >90% nonfluorescent form at pH
>> 6.2).
>
> Iain
>
> Iain Johnson Consulting
> Eugene, OR
>
>
>
> On Fri, Oct 25, 2013 at 2:12 PM, Cecile Chalouni
> <[hidden email]>wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi everybody,
>>
>> We are commonly using lysotracker to visualize acidic compartments which
>> includes different types of endocytic compartments in addition to the
>> lysosomes. I wonder if anybody knows what types of compartments are labeled
>> with the lysotracker or/and at what pH it starts emitting?
>>
>> Thank you in advance for your feedback!
>>
>> Best regards,
>>
>> Cecile
>>
>>
>> Cécile Chalouni, Ph. D
>> Genentech, Inc.
>> 1 DNA Way, Mail Stop 72b
>> So. San Francisco, California 94080
>>
>> p: 650-467-6772
>> c: 650-892-4763
>> e: [hidden email]
>>

Cécile Chalouni, Ph. D
Genentech, Inc.
1 DNA Way, Mail Stop 72b
So. San Francisco, California 94080

p: 650-467-6772
c: 650-892-4763
e: [hidden email]