fluorescein diacetate (FDA)

[F Javier Díez Guerra]

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Hi,

anybody knows if FDA (fluorescein diacetate)
distributes evenly within cells once incorporated
and cleaved by cellular esterases?

I assume that once cleaved, FDA can no longer
cross membranes and therefore remains trapped in soluble compartments.

In other words, can FDA be used as a marker of
internal cell volume due to its free diffusion
among continuous cellular compartments (Vg. cytosol + nuclear matrix)?

Thanks


F Javier Diez-Guerra, PhD
Profesor Titular
Centro de Biologia Molecular Severo Ochoa
C/ Nicolás Cabrera, 1
Universidad Autónoma
Ctra Colmenar Viejo Km 15
Cantoblanco, 28049 Madrid
SPAIN

phone:  [phone]
e-mail: [hidden email]

[Russell Spear]

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Hi

A problem with FDA it can also diffuse out of the cell after cleavage
causing the surrounding media to fluorese, fungal material diffuses in
cytosol, and leaves the nulear material less bright.

Russ

Russell N. Spear
Sr. Research Specialist
Dept. of Plant Pathology
Univ. of Wisconsin
1630 Linden Dr.
Madison WI 53706

voice 608.263.2093
fax     [phone]

>>> F Javier Diez Guerra <[hidden email]> 04/08/08 9:44 AM >>>
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal 

Hi,

anybody knows if FDA (fluorescein diacetate)
distributes evenly within cells once incorporated
and cleaved by cellular esterases?

I assume that once cleaved, FDA can no longer
cross membranes and therefore remains trapped in soluble compartments.

In other words, can FDA be used as a marker of
internal cell volume due to its free diffusion
among continuous cellular compartments (Vg. cytosol + nuclear matrix)?

Thanks


F Javier Diez-Guerra, PhD
Profesor Titular
Centro de Biologia Molecular Severo Ochoa
C/ Nicolás Cabrera, 1
Universidad Autónoma
Ctra Colmenar Viejo Km 15
Cantoblanco, 28049 Madrid
SPAIN

phone:  [phone]
e-mail: [hidden email]

[Rosemary.White]

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Yes, the cleaved product is moderately membrane-permeable over time,
especially if the cells are strongly loaded with the dye, but you can use
one of the larger, and less membrane-permeable relatives, such as CFDA -
there are others as well.

cheers,
Rosemary

Rosemary White                    [hidden email]
CSIRO Plant Industry            ph.     61 (0)2-6246 5475
GPO Box 1600                       fax.     [phone]
Canberra, ACT 2601
Australia



On 9/4/08 1:51 AM, "Russell Spear" <[hidden email]> wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hi
>
> A problem with FDA it can also diffuse out of the cell after cleavage
> causing the surrounding media to fluorese, fungal material diffuses in
> cytosol, and leaves the nulear material less bright.
>
> Russ
>
> Russell N. Spear
> Sr. Research Specialist
> Dept. of Plant Pathology
> Univ. of Wisconsin
> 1630 Linden Dr.
> Madison WI 53706
>
> voice 608.263.2093
> fax     [phone]
>
>>>> F Javier Diez Guerra <[hidden email]> 04/08/08 9:44 AM >>>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hi,
>
> anybody knows if FDA (fluorescein diacetate)
> distributes evenly within cells once incorporated
> and cleaved by cellular esterases?
>
> I assume that once cleaved, FDA can no longer
> cross membranes and therefore remains trapped in soluble compartments.
>
> In other words, can FDA be used as a marker of
> internal cell volume due to its free diffusion
> among continuous cellular compartments (Vg. cytosol + nuclear matrix)?
>
> Thanks
>
>
> F Javier Diez-Guerra, PhD
> Profesor Titular
> Centro de Biologia Molecular Severo Ochoa
> C/ Nicolás Cabrera, 1
> Universidad Autónoma
> Ctra Colmenar Viejo Km 15
> Cantoblanco, 28049 Madrid
> SPAIN
>
> phone:  [phone]
> e-mail: [hidden email]

[Jacqueline Ross]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi Javier,

I would suggest CMFDA. I've used it a lot for various cell types and can fix it if necessary. I find the nucleus often stains less intensely than the cytoplasm although it still labels up OK.

Kind regards,

Jacqui


Jacqueline Ross

Biomedical Imaging Microscopist
Biomedical Imaging Research Unit
School of Medical Sciences
Faculty of Medical & Health Sciences
The University of Auckland
Private Bag 92019
Auckland, NEW ZEALAND

Tel: [phone] Ext 87438
Fax: [phone]

http://www.health.auckland.ac.nz/biru/ 


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of F Javier Diez Guerra
Sent: Wednesday, 9 April 2008 2:44 a.m.
To: [hidden email]
Subject: fluorescein diacetate (FDA)

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi,

anybody knows if FDA (fluorescein diacetate) distributes evenly within cells once incorporated and cleaved by cellular esterases?

I assume that once cleaved, FDA can no longer cross membranes and therefore remains trapped in soluble compartments.

In other words, can FDA be used as a marker of internal cell volume due to its free diffusion among continuous cellular compartments (Vg. cytosol + nuclear matrix)?

Thanks


F Javier Diez-Guerra, PhD
Profesor Titular
Centro de Biologia Molecular Severo Ochoa C/ Nicolás Cabrera, 1 Universidad Autónoma Ctra Colmenar Viejo Km 15 Cantoblanco, 28049 Madrid SPAIN

phone:  [phone]
e-mail: [hidden email]

[Randy Learish-2]

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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

On this topic, I was wondering how well the chloro-methylated dyes are
retained.  The Probes handbook says these dyes have "mild" thiol-reactivity
which would explain why the Tracker dyes are better retained in living cells.  
But does that mean they actually cross-link to proteins?  If I lysed the cells
after labeling, would I see fluorescently labeled proteins on SDS-PAGE?

Randy

[John J. Lemasters]

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Calcein-AM is better FDA for labeling the cell interior. Calcein has 4
negative charges (maybe five) and is well retained. Also, calcein unlike
FDA is insensitive to physiological pH changes.

John

Randy Learish wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> On this topic, I was wondering how well the chloro-methylated dyes are
> retained.  The Probes handbook says these dyes have "mild" thiol-reactivity
> which would explain why the Tracker dyes are better retained in living cells.  
> But does that mean they actually cross-link to proteins?  If I lysed the cells
> after labeling, would I see fluorescently labeled proteins on SDS-PAGE?
>
> Randy
>
>
>  

--
John J. Lemasters, MD, PhD
Professor and South Carolina COEE Endowed Chair
Director, Center for Cell Death, Injury and Regeneration
Departments of Pharmaceutical Sciences and Biochemistry & Molecular Biology
Medical University of South Carolina
QF308 Quadrangle Building
280 Calhoun Street, MSC 140
Charleston, SC 29425

Office: 843-792-2153
Lab: 843-792-3530
Fax: [phone]
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