Membrane probe for CHO-K1 cells

[Kevin Sunley]

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Hello everyone,

I am currently working with a CHO-K1 cell line which we believe has
become particularly sensitive to mechanical stress.  We are attempting
to quantity the deformability of the cells when grown unattached in
suspension cultures following a method similar to Brown et al. (J.
Immunology (2001), 166:6640-6646).  Briefly, the cells are removed from
culture and adhered to coverslips, subject to centrifugal forces, fixed,
and the membrane shape is observed in X-Z or Y-Z profiles.

The protocol we're hoping to adapt used anti-CD45 mAb as a membrane
probe, however they were working with lymphocytes (mouse, if I remember
correctly).  Would anti-CD45 work on the CHO-K1 line as well, or does
anyone have any recommendations for a better membrane target?

Most likely the imaging will be done on an apotome-equipped Zeiss
AxioImager, so the target should be one that will provide a strong
enough signal for imaging, and hopefully one common enough that custom
mAbs aren't required.



Also, I just wanted to thank everyone who helped with suggestions for
getting our Ludl MAC controller working a few months ago.  It took a bit
of time but everything is up and working again now.

Thanks again,

Kevin Sunley
Department of Microbiology
University of Manitoba
Winnipeg, Canada

[mmodel]

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Kevin -

I would like to talk to you about this.

Michael Model, Ph.D.
Confocal Microscopy Core
Dpt. Biological Sciences
Kent State University
Kent, OH 44242
tel. [phone]


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Kevin Sunley
Sent: Thursday, May 01, 2008 2:32 PM
To: [hidden email]
Subject: Membrane probe for CHO-K1 cells

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

Hello everyone,

I am currently working with a CHO-K1 cell line which we believe has
become particularly sensitive to mechanical stress.  We are attempting
to quantity the deformability of the cells when grown unattached in
suspension cultures following a method similar to Brown et al. (J.
Immunology (2001), 166:6640-6646).  Briefly, the cells are removed from
culture and adhered to coverslips, subject to centrifugal forces, fixed,

and the membrane shape is observed in X-Z or Y-Z profiles.

The protocol we're hoping to adapt used anti-CD45 mAb as a membrane
probe, however they were working with lymphocytes (mouse, if I remember
correctly).  Would anti-CD45 work on the CHO-K1 line as well, or does
anyone have any recommendations for a better membrane target?

Most likely the imaging will be done on an apotome-equipped Zeiss
AxioImager, so the target should be one that will provide a strong
enough signal for imaging, and hopefully one common enough that custom
mAbs aren't required.



Also, I just wanted to thank everyone who helped with suggestions for
getting our Ludl MAC controller working a few months ago.  It took a bit

of time but everything is up and working again now.

Thanks again,

Kevin Sunley
Department of Microbiology
University of Manitoba
Winnipeg, Canada

[mmodel]

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

Sorry for sending the message to everyone

[Knecht, David]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal We express Gap43-GFP in cells when we want a membrane label.  Dave

On May 1, 2008, at 2:31 PM, Kevin Sunley wrote:

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

Hello everyone,

I am currently working with a CHO-K1 cell line which we believe has become particularly sensitive to mechanical stress.  We are attempting to quantity the deformability of the cells when grown unattached in suspension cultures following a method similar to Brown et al. (J. Immunology (2001), 166:6640-6646).  Briefly, the cells are removed from culture and adhered to coverslips, subject to centrifugal forces, fixed, and the membrane shape is observed in X-Z or Y-Z profiles.

The protocol we're hoping to adapt used anti-CD45 mAb as a membrane probe, however they were working with lymphocytes (mouse, if I remember correctly).  Would anti-CD45 work on the CHO-K1 line as well, or does anyone have any recommendations for a better membrane target?

Most likely the imaging will be done on an apotome-equipped Zeiss AxioImager, so the target should be one that will provide a strong enough signal for imaging, and hopefully one common enough that custom mAbs aren't required.



Also, I just wanted to thank everyone who helped with suggestions for getting our Ludl MAC controller working a few months ago.  It took a bit of time but everything is up and working again now.

Thanks again,

Kevin Sunley
Department of Microbiology
University of Manitoba
Winnipeg, Canada

Dr. David Knecht    

Department of Molecular and Cell Biology

Co-head Flow Cytometry and Confocal Microscopy Facility

U-3125

91 N. Eagleville Rd.

University of Connecticut

Storrs, CT 06269

860-486-2200

860-486-4331 (fax)

[Elijah-15]

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Dear all,

 

I've a small problem/technical issue and I'd be glad for any inputs.

 

I'm looking at two-photon autofluorescence for elastin. The system I'm using is as below:

 

Olympus FV300 coupled to IX71

Mira900 Ti:Sapph, 10W pump Verdi laser

Filters (epi-detection path): Chroma 700 shortpass, Schott glass filter 500nm Longpass filter.

 

The issue I have is that I don't seem to see the 2PF signals from elastin. I've used 790nm, 800nm, 830 nm (input power approx 90mW). I understand that the aforementioned wavelengths are typical for elastin 2PF. I do see autofluorescence with a 488nm Ar+ laser though. Any thoughts as to what's the issue here?

 

PS: How does one align the NIR beam properly into the objective and if it's not coming in straight, does this affect that epi-collected 2PF signals? I'm thinking that this is the issue, although I do get SHG signals from the collagen!

 

Thanks!

 

Elijah

 

  

[Chen Yuan Dong]

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Hi Elijah,

           Two-photon autofluorescence of elastin is fairly intense. If your system can pick up strong backward SHG from collagen, then you should be able to see elastin autofluorescence. Your system does need to be well aligned and you may want to check to make sure that your sample contains elastin. In the skin specimens we frequently use, sometimes you have to look around to find elastic fibers.

           Hope this helps.

 

Sincerely,

 

Chen

 

Chen Yuan Dong, PhD

Associate Professor

Department of Physics

National Taiwan University

Taipei 106, Taiwan, ROC

Office: Room 530

Tel: [phone]

Fax: [phone]

E-mail: [hidden email]