Membrane Staining
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I have a user who is working
with polarized epithelial cells and wants to label the basal-lateral membrane
with dye. She tried FM4-64 but it is not penetrating the tight junctions so it
is only labeling the apical surface. She wants to do this on live cells and
would prefer a dye to a fluorescent protein. Any suggestions would be helpful. Sincerely, Claire Claire
M. Brown, PhD Life
Sciences Complex Imaging Facility Director McGill
University Department of Biochemistry Montreal,
Quebec, H3G 0B1 http://www.lifesciencescomplex.mcgill.ca/imaging |
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Claire,
You didn't mention how the user is culturing the monolayer, but if suspended membranes (milliwell, transwell, etc) are being used, why not put the dye into the basal media? Andy At 08:13 AM 3/11/2009, you wrote: >I have a user who is working with polarized epithelial cells and >wants to label the basal-lateral membrane with dye. She tried FM4-64 >but it is not penetrating the tight junctions so it is only labeling >the apical surface. She wants to do this on live cells and would >prefer a dye to a fluorescent protein. Any suggestions would be helpful. > >Sincerely, > >Claire > >Claire M. Brown, PhD >Life Sciences Complex Imaging Facility Director >McGill University Department of Biochemistry >Montreal, Quebec, H3G 0B1 ><http://www.lifesciencescomplex.mcgill.ca/imaging>http://www.lifesciencescomplex.mcgill.ca/imaging > Andrew Resnick, Ph. D. Instructor Department of Physiology and Biophysics Case Western Reserve University 216-368-6899 (V) 216-368-4223 (F) |
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In reply to this post by Claire Brown
Hi Claire, just a thought, can
your researcher grow the polarized cells in a Transwell or similar insert? Any
dye would then have access to the basal surfaces… Jamie Barber www.vet.uga.edu/ID/tripp/personnel/Jamie From: Confocal Microscopy
List [mailto:[hidden email]] On Behalf Of Claire
Brown, Dr. I have a user who is working
with polarized epithelial cells and wants to label the basal-lateral membrane
with dye. She tried FM4-64 but it is not penetrating the tight junctions so it
is only labeling the apical surface. She wants to do this on live cells and
would prefer a dye to a fluorescent protein. Any suggestions would be helpful. Sincerely, Claire Claire
M. Brown, PhD Life
Sciences Complex Imaging Facility Director McGill
University Department of Biochemistry Montreal,
Quebec, H3G 0B1 http://www.lifesciencescomplex.mcgill.ca/imaging |
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Hi Claire,
We have had some good results using LavaCell from Fluorotechnics http://www.fluorotechnics.com/ It works well, sometimes not that bright unfortunately, but is very persistent. Cells still carry stain after 48 hours, and it is still only at the membrane. Cheers 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: +61 3 9341 3155 Mobile: +61422882700 Fax: +61 3 9341 3104 http://www.ludwig.edu.au/branch/research/platform/microscopy.htm ________________________________ From: Confocal Microscopy List on behalf of Jamie Barber Sent: Thu 12/03/2009 12:24 AM To: [hidden email] Subject: Re: Membrane Staining Hi Claire, just a thought, can your researcher grow the polarized cells in a Transwell or similar insert? Any dye would then have access to the basal surfaces... Jamie Barber AHRC Cell Imaging Facility Dept. of Infectious Diseases Univ. of Georgia, College of Veterinary Medicine 111 Carlton St. Bldg 1077 Athens GA 30602 Office: (706) 542-4092 Laboratory: (706) 542-9862 [hidden email] www.vet.uga.edu/ID/tripp/personnel/Jamie From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Claire Brown, Dr. Sent: Wednesday, March 11, 2009 9:13 AM To: [hidden email] Subject: Membrane Staining I have a user who is working with polarized epithelial cells and wants to label the basal-lateral membrane with dye. She tried FM4-64 but it is not penetrating the tight junctions so it is only labeling the apical surface. She wants to do this on live cells and would prefer a dye to a fluorescent protein. Any suggestions would be helpful. Sincerely, Claire Claire M. Brown, PhD Life Sciences Complex Imaging Facility Director McGill University Department of Biochemistry Montreal, Quebec, H3G 0B1 http://www.lifesciencescomplex.mcgill.ca/imaging <http://www.lifesciencescomplex.mcgill.ca/imaging> |
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Hi Cam, LavaCell is described as cell-permeable and interacting with proteins. How did you get it to stain only the membrane ? I would expect it to stain the protein on the membrane but also other protein in the cell. Cheers, JP Confocal Microscopy List <[hidden email]> wrote on 03/11/2009 12:40:53 PM: > Hi Claire, > We have had some good results using LavaCell from Fluorotechnics > > http://www.fluorotechnics.com/ > It works well, sometimes not that bright unfortunately, but is very > persistent. Cells still carry stain after 48 hours, and it is still > only at the membrane. > > Cheers > > 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: +61 3 9341 3155 > Mobile: +61422882700 > Fax: +61 3 9341 3104 > http://www.ludwig.edu.au/branch/research/platform/microscopy.htm > > ________________________________ > From: Confocal Microscopy List on behalf of Jamie Barber > Sent: Thu 12/03/2009 12:24 AM > To: [hidden email] > Subject: Re: Membrane Staining > > Hi Claire, just a thought, can your researcher grow the polarized > cells in a Transwell or similar insert? Any dye would then have > access to the basal surfaces... > > Jamie Barber > AHRC Cell Imaging Facility > Dept. of Infectious Diseases > Univ. of Georgia, College of Veterinary Medicine > 111 Carlton St. Bldg 1077 > Athens GA 30602 > Office: (706) 542-4092 > Laboratory: (706) 542-9862 > [hidden email] > www.vet.uga.edu/ID/tripp/personnel/Jamie > > From: Confocal Microscopy List [mailto:[hidden email]. > EDU] On Behalf Of Claire Brown, Dr. > Sent: Wednesday, March 11, 2009 9:13 AM > To: [hidden email] > Subject: Membrane Staining > > I have a user who is working with polarized epithelial cells and > wants to label the basal-lateral membrane with dye. She tried FM4-64 > but it is not penetrating the tight junctions so it is only labeling > the apical surface. She wants to do this on live cells and would > prefer a dye to a fluorescent protein. Any suggestions would be helpful. > > Sincerely, > > Claire > > Claire M. Brown, PhD > Life Sciences Complex Imaging Facility Director > McGill University Department of Biochemistry > Montreal, Quebec, H3G 0B1 > http://www.lifesciencescomplex.mcgill.ca/imaging <http://www. > lifesciencescomplex.mcgill.ca/imaging> |
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Hi JP,
You do see some staining in the cell (it is stronger when they are fixed). But, at least on the cells we have tried it on (LIM colon cancer lines), the membrane stain is much stronger than the cytoplasm stain. So when you capture an image the cytoplasm stain is virtually non existent due to the brightness of the membrane stain. The other advantage is that is works really well and is very persistent (its still there 48 hours later) in live cells. I have had proplems in the past with the FM dyes interalising into live cells very rapidly. Cheers Cam ________________________________ From: Confocal Microscopy List on behalf of Jean-Pierre CLAMME Sent: Fri 13/03/2009 4:08 AM To: [hidden email] Subject: Re: Membrane Staining Hi Cam, LavaCell is described as cell-permeable and interacting with proteins. How did you get it to stain only the membrane ? I would expect it to stain the protein on the membrane but also other protein in the cell. Cheers, JP Confocal Microscopy List <[hidden email]> wrote on 03/11/2009 12:40:53 PM: > Hi Claire, > We have had some good results using LavaCell from Fluorotechnics > > http://www.fluorotechnics.com/ > It works well, sometimes not that bright unfortunately, but is very > persistent. Cells still carry stain after 48 hours, and it is still > only at the membrane. > > Cheers > > 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: +61 3 9341 3155 > Mobile: +61422882700 > Fax: +61 3 9341 3104 > http://www.ludwig.edu.au/branch/research/platform/microscopy.htm > > ________________________________ > From: Confocal Microscopy List on behalf of Jamie Barber > Sent: Thu 12/03/2009 12:24 AM > To: [hidden email] > Subject: Re: Membrane Staining > > Hi Claire, just a thought, can your researcher grow the polarized > cells in a Transwell or similar insert? Any dye would then have > access to the basal surfaces... > > Jamie Barber > AHRC Cell Imaging Facility > Dept. of Infectious Diseases > Univ. of Georgia, College of Veterinary Medicine > 111 Carlton St. Bldg 1077 > Athens GA 30602 > Office: (706) 542-4092 > Laboratory: (706) 542-9862 > [hidden email] > www.vet.uga.edu/ID/tripp/personnel/Jamie > > From: Confocal Microscopy List [mailto:[hidden email]. > EDU] On Behalf Of Claire Brown, Dr. > Sent: Wednesday, March 11, 2009 9:13 AM > To: [hidden email] > Subject: Membrane Staining > > I have a user who is working with polarized epithelial cells and > wants to label the basal-lateral membrane with dye. She tried FM4-64 > but it is not penetrating the tight junctions so it is only labeling > the apical surface. She wants to do this on live cells and would > prefer a dye to a fluorescent protein. Any suggestions would be helpful. > > Sincerely, > > Claire > > Claire M. Brown, PhD > Life Sciences Complex Imaging Facility Director > McGill University Department of Biochemistry > Montreal, Quebec, H3G 0B1 > http://www.lifesciencescomplex.mcgill.ca/imaging <http://www. > lifesciencescomplex.mcgill.ca/imaging> |
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