Red Blood Cells Labelling

[Maria Calvo-2]

Dear all,

Does anyone know a good working method to label red blood cells with
FITC (or another dye) for intravital microscopy? We have extracted blood
and  tried to labell it with FITC without succeed. We need to follow
individual cells in the blood flow. I think that FITC labelling is the
most used and cheapest method. As far as I know, TRITC labelling is not
successful in this kind of cells and membrane labelling (such as
lectins) affects the rigidity of membrane.
Thank you in advance,

Maria Calvo

Dra. Maria Calvo

Unitat de Microscòpia Confocal
Serveis Cientificotècnics-C.Casanova
Facultat de Medicina
Universitat de Barcelona- IDIBAPS
C/ Casanova 143
Barcelona 08036

Tel: [phone]/39930
Fax: [phone]
E-mail: [hidden email]

[Cameron Nowell]

Hi Maria,
 
 
You may not need to lable them. RBCs are rather autofluorescent. You should be able to find an exitation range (somewhere around 750nm i would think) that will get them to light up by themsleves. No dye needed, you can't get more accurate or cheaper than that:)
 
Merry Christmas
 
 
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 Maria Calvo
Sent: Mon 21/12/2009 9:14 PM
To: [hidden email]
Subject: Red Blood Cells Labelling



Dear all,

Does anyone know a good working method to label red blood cells with
FITC (or another dye) for intravital microscopy? We have extracted blood
and  tried to labell it with FITC without succeed. We need to follow
individual cells in the blood flow. I think that FITC labelling is the
most used and cheapest method. As far as I know, TRITC labelling is not
successful in this kind of cells and membrane labelling (such as
lectins) affects the rigidity of membrane.
Thank you in advance,

Maria Calvo

Dra. Maria Calvo

Unitat de Microscòpia Confocal
Serveis Cientificotècnics-C.Casanova
Facultat de Medicina
Universitat de Barcelona- IDIBAPS
C/ Casanova 143
Barcelona 08036

Tel: [phone]/39930
Fax: [phone]
E-mail: [hidden email]

[Maria Calvo-2]

Dear Cameron,
Thanks for your help, we usually image RBCs by reflection. We want to
label only a pool of cells and inject them again to the blood because we
need to image individual cells.
Merry Christmas,
Maria

> Hi Maria,
>
>
> You may not need to lable them. RBCs are rather autofluorescent. You
> should be able to find an exitation range (somewhere around 750nm i would
> think) that will get them to light up by themsleves. No dye needed, you
> can't get more accurate or cheaper than that:)
>
> Merry Christmas
>
>
> 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 Maria Calvo
> Sent: Mon 21/12/2009 9:14 PM
> To: [hidden email]
> Subject: Red Blood Cells Labelling
>
>
>
> Dear all,
>
> Does anyone know a good working method to label red blood cells with
> FITC (or another dye) for intravital microscopy? We have extracted blood
> and  tried to labell it with FITC without succeed. We need to follow
> individual cells in the blood flow. I think that FITC labelling is the
> most used and cheapest method. As far as I know, TRITC labelling is not
> successful in this kind of cells and membrane labelling (such as
> lectins) affects the rigidity of membrane.
> Thank you in advance,
>
> Maria Calvo
>
> Dra. Maria Calvo
>
> Unitat de Microscòpia Confocal
> Serveis Cientificotècnics-C.Casanova
> Facultat de Medicina
> Universitat de Barcelona- IDIBAPS
> C/ Casanova 143
> Barcelona 08036
>
> Tel: [phone]/39930
> Fax: [phone]
> E-mail: [hidden email]
>
>

--
Dra. Maria Calvo

 Microscòpia Confocal. Unitat de Microscòpia
 Serveis Cientificotècnics-Campus Casanova
 Facultat de Medicina
 Universitat de Barcelona. IDIBAPS
 Casanova 143
 Barcelona 08036

 Tel: [phone]
 Fax: [phone]
 E-mail: [hidden email]

[George McNamara]

Hi Maria,

DiI. You can start with the paper below and figure out live mouse labeling yourself, or much faster, email the senior author (see http://www.nature.com/nprot/journal/v3/n11/abs/nprot.2008.172.html ), Professor Rong Wen, for his (not yet published) live mouse version.

... ahhh, just read your newest message about labeling a subset of cells (ex vivo) - use Rong's glucose saline::DiI in a test tube (probably less DiI than Rong injects).

Best wishes,

George
p.s. the original protocol is now very popular here at the U for labeling vessels (perfusion fixation and flushing the RBCs). DiI also fluoresces nicely when excited by multiphoton excitation.

Nat Protoc. 2008;3(11):1703-8.

Direct labeling and visualization of blood vessels with lipophilic carbocyanine dye DiI.

Li Y, Song Y, Zhao L, Gaidosh G, Laties AM, Wen R.

Bascom Palmer Eye Institute, Miller School of Medicine, University of Miami, 506 McKnight Building, 1638 NW 10th Avenue, Miami, Florida 33136, USA.

We describe a protocol to rapidly and reliably visualize blood vessels in experimental animals. Blood vessels are directly labeled by cardiac perfusion using a specially formulated aqueous solution containing 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), a lipophilic carbocyanine dye, which incorporates into endothelial cell membranes upon contact. By lateral diffusion, DiI also stains membrane structures, including angiogenic sprouts and pseudopodial processes that are not in direct contact. Tissues can be immediately examined by conventional and confocal fluorescence microscopy. High-quality serial optical sections using confocal microscopy are obtainable from thick tissue sections, especially at low magnification, for three-dimensional reconstruction. It takes less than 1 h to stain the vasculature in a whole animal. Compared with alternative techniques to visualize blood vessels, including space-occupying materials such as India ink or fluorescent dye-conjugated dextran, the corrosion casting technique, endothelial cell-specific markers and lectins, the present method simplifies the visualization of blood vessels and data analysis.

PMID: 18846097



At 05:14 AM 12/21/2009, you wrote:

Dear all,

Does anyone know a good working method to label red blood cells with
FITC (or another dye) for intravital microscopy? We have extracted blood
and  tried to labell it with FITC without succeed. We need to follow
individual cells in the blood flow. I think that FITC labelling is the
most used and cheapest method. As far as I know, TRITC labelling is not
successful in this kind of cells and membrane labelling (such as
lectins) affects the rigidity of membrane.
Thank you in advance,

Maria Calvo

Dra. Maria Calvo

Unitat de Microscòpia Confocal
Serveis Cientificotècnics-C.Casanova
Facultat de Medicina
Universitat de Barcelona- IDIBAPS
C/ Casanova 143
Barcelona 08036

Tel: [phone]/39930
Fax: [phone]
E-mail: [hidden email]

George McNamara, Ph.D.
Image Core Manager
Analytical Imaging Core Facility
University of Miami, Miller School of Medicine
Miami, FL 33136
[hidden email]
[hidden email]
305-243-8436 office
http://www.sylvester.org/AICF (Analytical Imaging Core Facility)
http://www.sylvester.org/AICF/pubspectra.zip (the entire 2000+ spectra .xlsx file is in the zip file)
http://home.earthlink.net/~geomcnamara

[Maria Calvo-2]

Dear George and Cameron,

Thanks for your answers. The list is very helpful. Finally RBC labelling
worked with FITC and we got very nice movies and measurements.
Merry Christmas

Maria Calvo

to all Cameron Nowell escribió:

> Hi Maria,
>  
> Ahh ok. What about something like CFSE? I have used it in the past to
> label stem cells from one mouse and reinject them into another mouse.
>  
>  
> Cheers
>  
> Cam
>  
>
> *From:* [hidden email] [mailto:[hidden email]]
> *Sent:* Tue 22/12/2009 9:02 AM
> *To:* Confocal Microscopy List
> *Cc:* Cameron Nowell
> *Subject:* Re: Red Blood Cells Labelling
>
> Dear Cameron,
> Thanks for your help, we usually image RBCs by reflection. We want to
> label only a pool of cells and inject them again to the blood because we
> need to image individual cells.
> Merry Christmas,
> Maria
>
> > Hi Maria,
> >
> >
> > You may not need to lable them. RBCs are rather autofluorescent. You
> > should be able to find an exitation range (somewhere around 750nm i
> would
> > think) that will get them to light up by themsleves. No dye needed, you
> > can't get more accurate or cheaper than that:)
> >
> > Merry Christmas
> >
> >
> > 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 Maria Calvo
> > Sent: Mon 21/12/2009 9:14 PM
> > To: [hidden email]
> > Subject: Red Blood Cells Labelling
> >
> >
> >
> > Dear all,
> >
> > Does anyone know a good working method to label red blood cells with
> > FITC (or another dye) for intravital microscopy? We have extracted blood
> > and  tried to labell it with FITC without succeed. We need to follow
> > individual cells in the blood flow. I think that FITC labelling is the
> > most used and cheapest method. As far as I know, TRITC labelling is not
> > successful in this kind of cells and membrane labelling (such as
> > lectins) affects the rigidity of membrane.
> > Thank you in advance,
> >
> > Maria Calvo
> >
> > Dra. Maria Calvo
> >
> > Unitat de Microscòpia Confocal
> > Serveis Cientificotècnics-C.Casanova
> > Facultat de Medicina
> > Universitat de Barcelona- IDIBAPS
> > C/ Casanova 143
> > Barcelona 08036
> >
> > Tel: [phone]/39930
> > Fax: [phone]
> > E-mail: [hidden email]
> >
> >
>
>
> --
> Dra. Maria Calvo
>
>  Microscòpia Confocal. Unitat de Microscòpia
>  Serveis Cientificotècnics-Campus Casanova
>  Facultat de Medicina
>  Universitat de Barcelona. IDIBAPS
>  Casanova 143
>  Barcelona 08036
>
>  Tel: [phone]
>  Fax: [phone]
>  E-mail: [hidden email]
>
>
> This communication is intended only for the named recipient and may
> contain information that is confidential, legally privileged or
> subject to copyright; the Ludwig Institute for Cancer Research Ltd
> does not waive any rights if you have received this communication in
> error.
> The views expressed in this communication are those of the sender and
> do not necessarily reflect the views of the Ludwig Institute for
> Cancer Research Ltd.

--


___________________________________

Dra. Maria Calvo

Unitat de Microscòpia Confocal
Serveis Cientificotècnics-C.Casanova
Facultat de Medicina
Universitat de Barcelona- IDIBAPS
C/ Casanova 143
Barcelona 08036

Tel: [phone]/39930
Fax: [phone]
E-mail: [hidden email]
___________________________________