CFSE LABEL

[Alejandra Quintana]

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I need an acute protocol to mark isolated hepatocytes (with CFSE) obtained by
collagenase digestion. I transplant isolated heptocyte to partial hepatectomized
(3/4) rat’s livers and I need to differentiate donor hepatocytes from recipient
ones. Isolated hepatocytes are obtained in Ringer solution. I proved with some
protocols in order to label the cells before transplant, but I failed. I obtained no
mark with confocal exploration. The marker is 5-(and-6)-Carboxyfluorescein
Diacetate, Succinimidyl Ester (5(6)-CFDA, SE; CFSE) - Mixed Isomers from
Invitrogen.  I recently bought the mark.  I transplant 150.000 hepatocytes /
ml.
If somebody has an appropriate and acute method to stain the cells berfore
transplant (with CFSE) I should be glad to received the protocol so as not to
lose time, mark and rats. Thank you very much.

Dra. Alejandra Beatriz Quintana
Area Morfología - Dto. de Ciencias Fisiológicas
Vice Directora de la Escuela de Bioquímica
Facultad de Ciencias Bioquímicas y Farmacéuticas
Suipacha 531/570 - (2000) ROSARIO
Universidad Nacional de Rosario
 Pcia. de Santa Fe - República Argentina

[mcammer]

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I would be surprised if CFSE were effective in the intact organ for more than a very brief time.
I've done imaging for researchers who have done transplant work such as you describe.  However, the markers were always protein expression in the donor cells not found in the recipient.  (In at least one of the studies this protein expression was also sufficient to restore partial organ function.)  

_________________________________________
Michael Cammer, Assistant Research Scientist
Skirball Institute of Biomolecular Medicine
Lab: [phone]  Cell: [phone]

________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Alejandra Quintana [[hidden email]]
Sent: Friday, August 03, 2012 10:22 PM
To: [hidden email]
Subject: CFSE LABEL

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I need an acute protocol to mark isolated hepatocytes (with CFSE) obtained by
collagenase digestion. I transplant isolated heptocyte to partial hepatectomized
(3/4) rat’s livers and I need to differentiate donor hepatocytes from recipient
ones. Isolated hepatocytes are obtained in Ringer solution. I proved with some
protocols in order to label the cells before transplant, but I failed. I obtained no
mark with confocal exploration. The marker is 5-(and-6)-Carboxyfluorescein
Diacetate, Succinimidyl Ester (5(6)-CFDA, SE; CFSE) - Mixed Isomers from
Invitrogen.  I recently bought the mark.  I transplant 150.000 hepatocytes /
ml.
If somebody has an appropriate and acute method to stain the cells berfore
transplant (with CFSE) I should be glad to received the protocol so as not to
lose time, mark and rats. Thank you very much.

Dra. Alejandra Beatriz Quintana
Area Morfología - Dto. de Ciencias Fisiológicas
Vice Directora de la Escuela de Bioquímica
Facultad de Ciencias Bioquímicas y Farmacéuticas
Suipacha 531/570 - (2000) ROSARIO
Universidad Nacional de Rosario
 Pcia. de Santa Fe - República Argentina

[Kilgore, Jason-2]

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** Vendor Response **

Dra. Quintana,

CFSE will work, but it will have some drawbacks (concentrations and times would need to be optimized, likely 10-20uM, 20-60min).  Due to dilution of intensity with each proliferation, the labeled cells will become undetectable from background within just a few days to a week.  The intensity and wavelength will likely not be as good for in vivo imaging, and the dye will likely be extracted upon doing any sectioning work, especially if using paraffin.

However, there have been some very successful similar tests using Qdot labeling (with Qtracker cell labeling reagents), which are very bright and photostable and are available in far red and near-IR emissions, good for in vivo imaging or getting around autofluorescence.  If you contact me offline, I would be happy to send you (or anyone else) a list of references where this has been done (particularly for labeling stem cells and injecting them in vivo).

Cheers,

Jason


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
www.invitrogen.com/technicalsupport

 




-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Alejandra Quintana
Sent: Friday, August 03, 2012 7:23 PM
To: [hidden email]
Subject: CFSE LABEL

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I need an acute protocol to mark isolated hepatocytes (with CFSE) obtained by
collagenase digestion. I transplant isolated heptocyte to partial hepatectomized
(3/4) rat's livers and I need to differentiate donor hepatocytes from recipient
ones. Isolated hepatocytes are obtained in Ringer solution. I proved with some
protocols in order to label the cells before transplant, but I failed. I obtained no
mark with confocal exploration. The marker is 5-(and-6)-Carboxyfluorescein
Diacetate, Succinimidyl Ester (5(6)-CFDA, SE; CFSE) - Mixed Isomers from
Invitrogen.  I recently bought the mark.  I transplant 150.000 hepatocytes /
ml.
If somebody has an appropriate and acute method to stain the cells berfore
transplant (with CFSE) I should be glad to received the protocol so as not to
lose time, mark and rats. Thank you very much.

Dra. Alejandra Beatriz Quintana
Area Morfología - Dto. de Ciencias Fisiológicas
Vice Directora de la Escuela de Bioquímica
Facultad de Ciencias Bioquímicas y Farmacéuticas
Suipacha 531/570 - (2000) ROSARIO
Universidad Nacional de Rosario
 Pcia. de Santa Fe - República Argentina

[Tommaso Mello]

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Dear Jason,

I am also interested in those references.
Besides, I have tried to visualize injected hepatocytes pre-loaded with
Qdots (celltracker kit QD705nm). I could see the Qdots in the fresh,
unfixed liver, but I was unable to find them once the tissue had been
FFPE.
Are you aware of issue with QD and paraffin embedding? Do you have
specific protocols for that?

Thank you in advance,

Best regards


Tommaso

Il giorno mar, 07/08/2012 alle 14.04 -0400, Kilgore, Jason ha scritto:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> ** Vendor Response **
>
> Dra. Quintana,
>
> CFSE will work, but it will have some drawbacks (concentrations and times would need to be optimized, likely 10-20uM, 20-60min).  Due to dilution of intensity with each proliferation, the labeled cells will become undetectable from background within just a few days to a week.  The intensity and wavelength will likely not be as good for in vivo imaging, and the dye will likely be extracted upon doing any sectioning work, especially if using paraffin.
>
> However, there have been some very successful similar tests using Qdot labeling (with Qtracker cell labeling reagents), which are very bright and photostable and are available in far red and near-IR emissions, good for in vivo imaging or getting around autofluorescence.  If you contact me offline, I would be happy to send you (or anyone else) a list of references where this has been done (particularly for labeling stem cells and injecting them in vivo).
>
> Cheers,
>
> Jason
>
>
> 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
> www.invitrogen.com/technicalsupport
>
>  
>
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Alejandra Quintana
> Sent: Friday, August 03, 2012 7:23 PM
> To: [hidden email]
> Subject: CFSE LABEL
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I need an acute protocol to mark isolated hepatocytes (with CFSE) obtained by
> collagenase digestion. I transplant isolated heptocyte to partial hepatectomized
> (3/4) rat's livers and I need to differentiate donor hepatocytes from recipient
> ones. Isolated hepatocytes are obtained in Ringer solution. I proved with some
> protocols in order to label the cells before transplant, but I failed. I obtained no
> mark with confocal exploration. The marker is 5-(and-6)-Carboxyfluorescein
> Diacetate, Succinimidyl Ester (5(6)-CFDA, SE; CFSE) - Mixed Isomers from
> Invitrogen.  I recently bought the mark.  I transplant 150.000 hepatocytes /
> ml.
> If somebody has an appropriate and acute method to stain the cells berfore
> transplant (with CFSE) I should be glad to received the protocol so as not to
> lose time, mark and rats. Thank you very much.
>
> Dra. Alejandra Beatriz Quintana
> Area Morfología - Dto. de Ciencias Fisiológicas
> Vice Directora de la Escuela de Bioquímica
> Facultad de Ciencias Bioquímicas y Farmacéuticas
> Suipacha 531/570 - (2000) ROSARIO
> Universidad Nacional de Rosario
>  Pcia. de Santa Fe - República Argentina

--
__________________

Tommaso Mello, PhD

Gastroenterology Unit
Dept. of Clinical Pathophysiology
University of Florence
Viale Pieraccini, 6
50139 - FIRENZE, ITALY
Phone:  [phone]
FAX:    [phone]