imaging cells on transwell membranes
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Search the CONFOCAL archive at
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A new confocal user came to me just now and asked if we
could image cells grown on transwell membranes. I sputtered a bit and then
said I would pose the question here. The advantage to the user is that the cells are more
polarized when they use the transwells. The cells would grow on the
inside membrane, they are not expecting them to migrate to the bottom (outside)
part of the membrane. They would fix the cells and then do the immuno labeling
on the cells while they are still attached to the membrane. We have an upright Zeiss 510META confocal. I know from experience in our histology lab that if you cut
out the membrane it tends to roll up like a scroll. Any ideas/suggestions? Doug ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ Douglas W. Cromey, M.S. - Assistant Scientific Investigator Dept. of Cell Biology & Anatomy, University of Arizona 1501 N. Campbell Ave, Tucson, AZ 85724-5044 USA office: AHSC
4212 email: [hidden email] voice:
520-626-2824 fax: 520-626-2097 http://swehsc.pharmacy.arizona.edu/exppath/ Home of: "Microscopy and Imaging Resources on the
WWW" |
 
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Andrew Resnick |
Re: imaging cells on transwell membranes
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal This is exactly like the setup I use- suspended filters and an upright microscope. Live cell imaging is easy (dipping objectives), fixed samples requires a little prep. Here's how we remove the filters, the whole process takes about 5 minutes once you are practiced: Place the transwell/milliwell filter insert onto a glass slide, right side up. There should be a little fluid on the top surface, and a liquid film between the filter and slide. If using a milliwell insert, orient one of the little feet at 12:00. Using a scalpel and holding the insert flat against the glass, make two cuts along the boundary- one from about 1:00 to 6:00, the other from 11:00 to 6:00. The fluid helps keep the filter flat, but if the edges roll up, that's fine for now. Slightly tilt the insert towards you- the point of attachment of the filter should be slightly off the glass. Use a small pair of scissors to cut the filter still attached to the insert. I've used a Noyes scissor, a Barraquer scissor, and a Vannas scissor. I prefer the Barraquer because I'm a lefty and the angle works better, but any of them are ok as long as they are SHARP. Now the filter should be free- remove the insert, and use a couple of probes to flatten out the filter. If the filter does not come free, carefully cut away wherever the filter is still attached with the scissors. Carefully wick off the fluid with a Kimwipe or whatever, add Vectashield and a glass coverslip. Seal the edges with nail polish, and you are good to go. Some people build up a spacer, but my experience is that it's not required. Let me know if you need additional details. Andy At 12:14 PM 5/13/2008, you wrote: >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >A new confocal user came to me just now and asked if we could image >cells grown on transwell membranes. I sputtered a bit and then said >I would pose the question here. > >The advantage to the user is that the cells are more polarized when >they use the transwells. The cells would grow on the inside >membrane, they are not expecting them to migrate to the bottom >(outside) part of the membrane. They would fix the cells and then >do the immuno labeling on the cells while they are still attached to >the membrane. > >We have an upright Zeiss 510META confocal. > >I know from experience in our histology lab that if you cut out the >membrane it tends to roll up like a scroll. > >Any ideas/suggestions? > >Doug > >^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ >Douglas W. Cromey, M.S. - Assistant Scientific Investigator >Dept. of Cell Biology & Anatomy, University of Arizona >1501 N. Campbell Ave, Tucson, AZ 85724-5044 USA > >office: AHSC 4212 email: [hidden email] >voice: 520-626-2824 fax: 520-626-2097 > >http://swehsc.pharmacy.arizona.edu/exppath/ >Home of: "Microscopy and Imaging Resources on the WWW" > 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 cromey
Search the CONFOCAL archive at
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Doug
Bioptechs has a product and technique that addresses this problem. We have many users of this technique. We plate our cells on the basal surface of the transwell insert in the following manner. 1. A bio-compatible piece of tubing is cut into a small cylinder of suitable geometry as to provide a shallow well and fluid barrier when placed over the distal end of the insert. 2. A plug made of silicon is placed into the tubular portion of the insert to prevent leakage through the membrane. (not always necessary) 3. The insert is inverted (membrane side up) and cells are poured into the well defined by the cylindrical tubing surrounding the membrane. 4. The insert/s with cells are returned to the incubator and allowed to plate in this inverted orientation. 5. After the cells have plated, the plug and tubing are removed from the insert and the insert is returned to the original tray right side up with appropriate media and allowed to divide until confluent. 6. When the cells are confluent the insert is placed into a Bioptechs Delta T4 Transwell Adapter which is then lowered into a Delta T dish on the microscope. The cells are now facing down for easy imaging through the coverglass bottomed self-heating Delta T dish. See the Bioptechs web site for details. 7. Cells on the membrane can be perfused on either the apical or basal surface with appropriate accessory during microscopy. Dan On May 13, 2008, at 1:14 PM, Doug Cromey wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal A new confocal user came to me just now and asked if we could image cells grown on transwell membranes. I sputtered a bit and then said I would pose the question here. The advantage to the user is that the cells are more polarized when they use the transwells. The cells would grow on the inside membrane, they are not expecting them to migrate to the bottom (outside) part of the membrane. They would fix the cells and then do the immuno labeling on the cells while they are still attached to the membrane. We have an upright Zeiss 510META confocal. I know from experience in our histology lab that if you cut out the membrane it tends to roll up like a scroll. Any ideas/suggestions? Doug ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ Douglas W. Cromey, M.S. - Assistant Scientific Investigator Dept. of Cell Biology & Anatomy, University of Arizona 1501 N. Campbell Ave, Tucson, AZ 85724-5044 USA office: AHSC 4212 email: [hidden email] voice: 520-626-2824 fax: 520-626-2097 Home of: "Microscopy and Imaging Resources on the WWW" |
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G. Esteban Fernandez |
Re: imaging cells on transwell membranes
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In reply to this post by cromey
Search the CONFOCAL archive at
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We do this all the time in our Core facility (we too have a 510 META, inverted). Users fix and stain the cells on the membrane, then I decant their final wash buffer and cut out the wet membrane with a razor blade while holding it with forceps. I've never had one roll up on me. I put the moist membrane onto a slide, add mount, and cover.
Esteban
On Tue, May 13, 2008 at 12:14 PM, Doug Cromey <[hidden email]> wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal -- G. Esteban Fernandez, Ph.D. Associate Director Molecular Cytology Core Facility University of Missouri 120 Bond Life Sciences Center Columbia, MO 65211 http://www.biotech.missouri.edu/mcc/ 573-882-4895 573-884-9676 fax |
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Jacqueline Ross |
Re: imaging cells on transwell membranes
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hi Doug,
I've used these transwell membranes before quite a bit. Depending on the
membrane, I have found that they do have a tendency to roll up and are awkward
to work with when using the very small ones, however the main thing is to hold
the membrane firmly when cutting it out as Esteban mentioned, so that you
don't lose the orientation and end up mounting it upside
down.
Once you have the mounting medium on it, you can then use the coverslip
to weigh it down and it generally flattens down OK.
Kind regards,
Jacqui
Jacqueline Ross
Biomedical Imaging
Microscopist From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of G. Esteban Fernandez Sent: Wednesday, 14 May 2008 9:42 a.m. To: [hidden email] Subject: Re: imaging cells on transwell membranes We do this all the time in our Core facility (we too have a 510 META,
inverted). Users fix and stain the cells on the membrane,
then I decant their final wash buffer and cut out the wet membrane with a
razor blade while holding it with forceps. I've never had one roll up
on me. I put the moist membrane onto a slide, add mount, and cover.
Esteban
On Tue, May 13, 2008 at 12:14 PM, Doug Cromey <[hidden email]> wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal -- G. Esteban Fernandez, Ph.D. Associate Director Molecular Cytology Core Facility University of Missouri 120 Bond Life Sciences Center Columbia, MO 65211 http://www.biotech.missouri.edu/mcc/ 573-882-4895 573-884-9676 fax |
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Kevin Conway |
Re: imaging cells on transwell membranes
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In reply to this post by cromey
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Doug- The scrolling of the transwell membrane isn't necessarily a bad thing, admittedly it depends what you are looking at. My wife and I came up with a way of rolling up her membranes on which she had grown organotypic rat epidermal cells - Dale Laird's lab at Univ. Western Ontario would be the people to contact now. The stratified epithelium tolerated the rolling well, we just froze it in OCT and cryosectioned transversely at around 8-10 um and the images were quite good. Sorry you didn't mention what it was the polarized epithelium was. With a nonstratified epithelium your mileage may vary (significantly). Cheers, Kevin Kevin Conway Imaging Specialist Nikon Canada, Inc [hidden email] |
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Renato A. Mortara |
405 diode laser on a Zeiss 510 Meta
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In reply to this post by Andrew Resnick
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi, I wonder if anyone has a 405 diode laser installed on a Zeiss 510 Meta. Any feedbacks (specifically, how well imaging of e.g. DAPI can be done with this laser) would be grately appreciated ! Many thanks, Renato Renato A. Mortara Parasitology Division UNIFESP - Escola Paulista de Medicina Rua Botucatu, 862, 6th floor São Paulo, SP 04023-062 Brazil Phone: 55 11 5579-8306 Fax: 55 11 5571-1095 email: [hidden email] home page: www.ecb.epm.br/~ramortara |
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Csúcs Gábor |
Re: 405 diode laser on a Zeiss 510 Meta
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hallo Renato, We have it. It works nicely for DAPI. Cheers Gabor |
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Ian Montgomery |
Re: 405 diode laser on a Zeiss 510 Meta
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In reply to this post by Renato A. Mortara
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal 405 laser works beautifully for DAPI. Dr. Ian Montgomery, Histotechnology, I.B.L.S. Support Unit, Thomson Building, University of Glasgow, Glasgow, G12 8QQ. -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Renato Mortara Sent: 28 August 2008 13:34 To: [hidden email] Subject: 405 diode laser on a Zeiss 510 Meta Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi, I wonder if anyone has a 405 diode laser installed on a Zeiss 510 Meta. Any feedbacks (specifically, how well imaging of e.g. DAPI can be done with this laser) would be grately appreciated ! Many thanks, Renato Renato A. Mortara Parasitology Division UNIFESP - Escola Paulista de Medicina Rua Botucatu, 862, 6th floor São Paulo, SP 04023-062 Brazil Phone: 55 11 5579-8306 Fax: 55 11 5571-1095 email: [hidden email] home page: www.ecb.epm.br/~ramortara |
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William A. Monroe |
Re: 405 diode laser on a Zeiss 510 Meta
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In reply to this post by Renato A. Mortara
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Greetings, We have the 405 diode laser on a Zeiss 510 (without Meta) and it works quite well for DAPI. Best Wishes, Bill Monroe ********************* >>> Renato Mortara <[hidden email]> 08/28/08 7:34 AM >>> Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi- bin/wa?S1=confocal Hi, I wonder if anyone has a 405 diode laser installed on a Zeiss 510 Meta. Any feedbacks (specifically, how well imaging of e.g. DAPI can be done with this laser) would be grately appreciated ! Many thanks, Renato Renato A. Mortara Parasitology Division UNIFESP - Escola Paulista de Medicina Rua Botucatu, 862, 6th floor São Paulo, SP 04023- 062 Brazil Phone: 55 11 5579- 8306 Fax: 55 11 5571- 1095 email: [hidden email] home page: www.ecb.epm.br/~ramortara |
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Craig Brideau |
Re: 405 diode laser on a Zeiss 510 Meta
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Search the CONFOCAL archive at
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Don't know about Zeiss, but we used a 405 Diode laser on a Nikon C1Si with DAPI and got good images.
Craig On Thu, Aug 28, 2008 at 7:56 AM, William A. Monroe <[hidden email]> wrote: Greetings, |
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Richard Harris-6 |
Re: 405 diode laser on a Zeiss 510 Meta
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In reply to this post by Renato A. Mortara
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal We've a new Zeiss 510 Dou system and the 405 works really well with DAPI Rick, Richard Harris, Manager - Imaging and Data Systems The Biotron - Experimental Climate Change Research University of Western Ontario, London Ontario, CANADA. N6A 5B7 Ph. 519-661-2111 ext. 86780 Fax 519-661-3935 e-mail [hidden email] web: www.biotron.uwo.ca -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Renato Mortara Sent: Thursday, August 28, 2008 8:34 AM To: [hidden email] Subject: 405 diode laser on a Zeiss 510 Meta Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi, I wonder if anyone has a 405 diode laser installed on a Zeiss 510 Meta. Any feedbacks (specifically, how well imaging of e.g. DAPI can be done with this laser) would be grately appreciated ! Many thanks, Renato Renato A. Mortara Parasitology Division UNIFESP - Escola Paulista de Medicina Rua Botucatu, 862, 6th floor São Paulo, SP 04023-062 Brazil Phone: 55 11 5579-8306 Fax: 55 11 5571-1095 email: [hidden email] home page: www.ecb.epm.br/~ramortara |
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In reply to this post by Renato A. Mortara
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Renato, we have one on a 510 Meta. It works well for DAPI (and Hoechst) especially on cultured cells. On tissue sections, we have sometimes had less than perfect imaging. This is propably due to the fact that as 405 is a rather inefficient exciter of DAPI, we have to increase laser power and or detector gain more than what would be needed for UV excitation. This in turn increases tissue autofluorescence in some types of fixed samples. Best regards, Jan Grawé Jan Grawé Cell Analysis Core Facility Rudbecklaboratoriet/C5 Dag hammarskjölds väg 20 SE-75185 Uppsala SWEDEN Phone: +(0)18-4714656 Cell: +(0)70-2577874 [hidden email] www.rudbeck.uu.se/cellanalys |
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Renato A. Mortara |
RES: 405 diode laser on a Zeiss 510 Meta
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In reply to this post by Richard Harris-6
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Thanks Rick !! Best wishes Renato -----Mensagem original----- De: Confocal Microscopy List [mailto:[hidden email]] Em nome de Richard Harris Enviada em: quinta-feira, 28 de agosto de 2008 21:38 Para: [hidden email] Assunto: Re: 405 diode laser on a Zeiss 510 Meta Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal We've a new Zeiss 510 Dou system and the 405 works really well with DAPI Rick, Richard Harris, Manager - Imaging and Data Systems The Biotron - Experimental Climate Change Research University of Western Ontario, London Ontario, CANADA. N6A 5B7 Ph. 519-661-2111 ext. 86780 Fax 519-661-3935 e-mail [hidden email] web: www.biotron.uwo.ca -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Renato Mortara Sent: Thursday, August 28, 2008 8:34 AM To: [hidden email] Subject: 405 diode laser on a Zeiss 510 Meta Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi, I wonder if anyone has a 405 diode laser installed on a Zeiss 510 Meta. Any feedbacks (specifically, how well imaging of e.g. DAPI can be done with this laser) would be grately appreciated ! Many thanks, Renato Renato A. Mortara Parasitology Division UNIFESP - Escola Paulista de Medicina Rua Botucatu, 862, 6th floor São Paulo, SP 04023-062 Brazil Phone: 55 11 5579-8306 Fax: 55 11 5571-1095 email: [hidden email] home page: www.ecb.epm.br/~ramortara No virus found in this incoming message. Checked by AVG - http://www.avg.com Version: 8.0.138 / Virus Database: 270.6.11/1639 - Release Date: 28/8/2008 07:39 |
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Alice L. Givan |
Re: 405 diode laser on a Zeiss 510 Meta
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In reply to this post by Renato A. Mortara
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal We have a 405nm laser on our Zeiss 510 Meta system (NOT MP). The 405 line works fine -- but we have never been able to get the fluorescence from the 405 excitation lined up correctly (automatically) with the fluorescence from the other (VIS) lasers. This problem has haunted us (and our Zeiss engineers) ever since we purchased the instrument. Alice Alice L. Givan, Director Englert Cell Analysis Laboratory of the Norris Cotton Cancer Center Dartmouth Medical School Lebanon, NH 03756 USA tel 603-650-7661 fax 603-650-6130 [hidden email] www.dartmouth.edu/~celllab |
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