indicator for membrane damage in live cells.

[Aryeh Weiss]

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Does anyone has suggestions for indicators of membrane damage (possibly
short lived)  in live cells.

We are trying propidium iodide, which is not supposed to enter intact
live cells, but may enter a damaged transiently damaged membrane and
then light up in the nucleus.

The nice thing about  PI is that it is not fluorescent until it
associates with DNA or RNA.

Are there other methods that people on the list can suggest?

Thanks in advance.

Best regards
--aryeh

--
Aryeh Weiss
Faculty of Engineering
Bar Ilan University
Ramat Gan 52900 Israel

Ph:  972-3-5317638
FAX: [phone]

[F Javier Diez Guerra]

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Dear Aryeh,

I am not sure if this could help.

Not exactly an indicator on plasma membrane damage, but of stress and
loss of homeostasis is that phosphatidylserine is externalized to the
outer leaflet of the plasma membrane. This can be easily visualized with
Annexin V conjugated to a fluorophore (there are many commercial sources
of this reagent). This happens before PI can get into the cells and
label DNA.

Best

Javier


El 21/05/2021 a las 14:28, Aryeh Weiss escribió:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Does anyone has suggestions for indicators of membrane damage
> (possibly short lived)  in live cells.
>
> We are trying propidium iodide, which is not supposed to enter intact
> live cells, but may enter a damaged transiently damaged membrane and
> then light up in the nucleus.
>
> The nice thing about  PI is that it is not fluorescent until it
> associates with DNA or RNA.
>
> Are there other methods that people on the list can suggest?
>
> Thanks in advance.
>
> Best regards
> --aryeh
>

[Lemasters, John J.]

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Hi Aryeh,

Load cells with calcein using calcein-AM. Calcein leaks out with membrane damage. This is the basis for commercial live-dead assays (probably ThermoFisher)  using calcien in combination with propidium iodide.

Best, John

--
John J. Lemasters, MD, PhD
Professor and GlaxoSmithKline Distinguished Endowed Chair
Director, Center for Cell Death, Injury & Regeneration
Departments of Drug Discovery & Biomedical Sciences and Biochemistry & Molecular Biology
Medical University of South Carolina
DD504 Drug Discovery Building
70 President Street, MSC 139
Charleston, SC 29425

Office: 843-876-2360
Lab: 843-876-2354
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Email: [hidden email]
https://education.musc.edu/MUSCApps/FacultyDirectory/Lemasters-John

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Aryeh Weiss
Sent: Friday, May 21, 2021 8:29 AM
To: [hidden email]
Subject: indicator for membrane damage in live cells.

CAUTION: External

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Does anyone has suggestions for indicators of membrane damage (possibly short lived)  in live cells.

We are trying propidium iodide, which is not supposed to enter intact live cells, but may enter a damaged transiently damaged membrane and then light up in the nucleus.

The nice thing about  PI is that it is not fluorescent until it associates with DNA or RNA.

Are there other methods that people on the list can suggest?

Thanks in advance.

Best regards
--aryeh

--
Aryeh Weiss
Faculty of Engineering
Bar Ilan University
Ramat Gan 52900 Israel

Ph:  972-3-5317638
FAX: [phone]

[Christian Kukat]

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Dear Aryeh,

In flow cytometry, many dyes such as PI are used as viablity dyes.
Examples are, next to PI, DAPI, 7-AAD, SYTOX, DRAQ7, etc.

There are also fixable viability dyes from many manufacturers.

And as mentioned by John, dyes such as Calcein-AM can be used as vital dyes, to determine which cells are still metabolically active.

One example for a publication mentioning this is:
Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition)
by Cossarizza et al., 2019 in Eur. J. Immunol.

Chapter 3.4 Dead cell exclusion, cell viability, and sample freezing
https://doi.org/10.1002/eji.201970107 <https://doi.org/10.1002/eji.201970107>


Best wishes,

Christian

_______________________________________________________________________

Christian Kukat, PhD
Head of FACS & Imaging Core Facility
Max Planck Institute for Biology of Ageing

Joseph-Stelzmann-Str. 9b, D-50931 Köln / Cologne, Germany

E-mail: [hidden email]
www.age.mpg.de
_______________________________________________________________________

> On 21. May 2021, at 14:28, Aryeh Weiss <[hidden email]> wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Does anyone has suggestions for indicators of membrane damage (possibly short lived)  in live cells.
>
> We are trying propidium iodide, which is not supposed to enter intact live cells, but may enter a damaged transiently damaged membrane and then light up in the nucleus.
>
> The nice thing about  PI is that it is not fluorescent until it associates with DNA or RNA.
>
> Are there other methods that people on the list can suggest?
>
> Thanks in advance.
>
> Best regards
> --aryeh
>
> --
> Aryeh Weiss
> Faculty of Engineering
> Bar Ilan University
> Ramat Gan 52900 Israel
>
> Ph:  972-3-5317638
> FAX: [phone]

[Jyoti Jaiswal]

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FM dyes also work as PI and other DNA binding dyes, but leaves nucleic acid untouched. Their fluorescence increases upon entering cells and binding intracellular membranes and the labeling intensity is proportional to extent of damage. Here is our description of its use - https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4089398/.

Jyoti

[Aryeh Weiss]

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I want to thank the many list members who replied both on  and off list.
We received many suggestions, including using calcium indicators, a
variety of suggestions for anionic dyes that do not enter intact cells,
many DNA indicators in case we do not like PI, references on the use of
PI for this purpose, and references from people who have done similar
studies.
So we have a lot to chew on , and I can say that  just about anything I
might do with a scope has been done by someone (or many someones) on
this list.

Best regards
--aryeh

On 23/05/2021 21:28, Jyoti Jaiswal wrote:
> *****
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> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> FM dyes also work as PI and other DNA binding dyes, but leaves nucleic acid untouched. Their fluorescence increases upon entering cells and binding intracellular membranes and the labeling intensity is proportional to extent of damage. Here is our description of its use - https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4089398/.
>
> Jyoti


--
Aryeh Weiss
Faculty of Engineering
Bar Ilan University
Ramat Gan 52900 Israel

Ph:  972-3-5317638
FAX: [phone]