mounting suspension cells
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Last week I had some users bring in suspension cells for me
to image with our confocal. Darn things were moving so much that it was
difficult to get two colors to line up using sequential scanning. Apparently
these are non-adherent cells, so growing them on a coverslip is not an option.
Any suggestions? What they gave me was a drop of cells in culture media,
we put it between a slide and coverslip. 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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Franco Del Principe |
Re: mounting suspension cells
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Doug
Two things come to mind: 1) Did you use plasticine "feet" on the coverslip? When using square coverslips you can streak each corner over a piece of plasticine leaving a bit of it attached to the corner of the glass. This gives the coverslip a support to prevent squeezing of the liquid and subsequent moving of the trapped medium underneath. It is quite useful when operating with immersion oil. 2) Some cell types' adherence capacity is temperature dependent. HEK293 for instance detach as soon they come out of the 37 degree incubator. Cheers Franco [hidden email] wrote: > Last week I had some users bring in suspension cells for me to image > with our confocal. Darn things were moving so much that it was > difficult to get two colors to line up using sequential scanning. > Apparently these are non-adherent cells, so growing them on a coverslip > is not an option. Any suggestions? What they gave me was a drop of > cells in culture media, we put it between a slide and coverslip. > > > > 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" > > > > > ------------------------------------------------------------------------ > > > No virus found in this incoming message. > Checked by AVG - http://www.avg.com > Version: 8.0.173 / Virus Database: 270.7.6/1710 - Release Date: 10/6/2008 9:23 AM > -- ___ ___ _______ / / / / / ____/ / / / / / /___ / / / / /___ / / /___ / / ____/ / /______/ /__/ /______/ Dr. Franco Del Principe ------------------------------------------------------------ LIFE IMAGING SERVICES GmbH Fon +41 61 711 6461 Efringerstrasse 79 Fax +41 61 711 6462 CH-4057 Basel, Switzerland Mob +41 79 672 4694 E-mail [hidden email] Web http://www.lis.ch |
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In reply to this post by cromey
Hi Doug,
we work with two lines of suspension cells and freshly isolated cells. I've been successful with live (FURA-2 and GFP) and fixed cell imaging of cells attached to coverslips coated with poly-L-lysine or BD's Cell Tak. There are many protocols for poly-l-lysine coating. I immerse sterilized coverslips for 1 hour at 37degC in a 0.5-1mg/ml poly-L-lysine solution followed by three washes with ddH2O and one wash with serum-free culture medium. I apply cells after that and let them adhere overnight. For Cell Tak, I spread four microliters of the delivered, neat solution onto a coverslip using a rubber spatula. I then let the coverslip dry under a bright lamp, wash the coverslip once with ddH2O, and then let it dry again (I'll usually aspirate off as much water as I can before the final drying). Then, I place half a milliliter droplet of cell suspension on the coverslip and let the cells attach for 20 minutes to an hour at 37degC. It's also possible to lay down Cell Tak by adsorption too, but my applications haven't required it. For our cells (mast cell lines), the faster, but more expensive, Cell Tak option usually results in more attached cells. Best of Luck, Nate -- Nathan O'Connor Silver Laboratory Department of Physiology and Biophysics Weill Cornell Medical College New York, NY 10021 On Mon, Oct 6, 2008 at 11:36 AM, <[hidden email]> wrote:
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Aryeh Weiss |
parfocality problem with LWD objectives solved.
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The problem I asked about a couple of weeks ago (inability to image with LWD
objectives in a IX81) is solved. As expected, it was something very simple. Turns out that the LUDL stage on that system is indeed high, as I noticed. What I did not see the first time is that the objectives that came with the system each have a brass extension ring into which they thread, and which adds a few mm to their height. I assume that this was done to resolve some mechanical issue with the stage, though I am not sure. I think the rest is clear. Today I got a closer look at the objectives, and had that aha! moment. Placement of the ring on my LWD objective solved the problem. Thanks to list members to wrote to me with various suggestions. --aryeh -- Aryeh Weiss School of Engineering Bar Ilan University Ramat Gan 52900 Israel Ph: 972-3-5317638 FAX: 972-3-7384050 |
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In reply to this post by cromey
Doug,
You did not mention exactly what you were trying to image in/on the cells. Cells that have been developed to grow in suspension have been selected for this ability and they do not generally have the ECM/integrin/cytoskeletal network that provides surface adhesion. Then, of course, someone may have simply given you some freshly trypsinized cells resulting in the same problem, namely the lack of surface adhesion molecules. In any case, if the cells do not have to be imaged live, cells can be gently pelleted and washed a couple of times with iso-osmotic PBS to eliminate extraneous proteins from growth medium. After a couple of rinses the cells can be suspended then fixed with the usual reagents say 2% paraformaldehyde in PBS. After 30 minutes to an hour the cells will usually be sufficiently permeabilized to allow labeling with Ab fluorophore conjugates, etc. Conventional labeling on coverslips or slides is easily adapted to perform in suspension using gentle pelleting and resuspension for rinsing cells. As for mounting in something that is ~ physiological, I like to use a solution of 50% glycerol plus PBS. The glycerol viscosity helps keep cells from moving around (more glycerol helps, too), and because of the lower dielectric constant, the electrostatic binding to coverslips or slides treated with polylysine also helps with adhesion (must have M.W. 300 kDa). Even better would be to make some aminopropyl silane treated slides and coverslips. Polylysine can come off the glass and wrap cells together in the extreme. The aminopropyl silane treatment creates covalently bound positively charged surfaces that won't come off (the amino group can become oxidized but takes a very long time). Although I have not tried this with live cells, aminopropyl glass would be first thing I'd try. However, there is a potential problem with using anything that creates a very large electrostatic field in that this could have a strong effect on the PM causing channels to open/close, local lipid rearrangement (lipid flip-flop), and generally altered kinetics of membrane transporters and receptors. Cell Tak as Nathan suggested could prove advantageous for live cells; however, some of the same problems could apply in that anything you do to cells that are without the capacity to adhere are likely to experience significant perturbation in their cell surfaces. Further, I also have a concern about Cell Tak in that its crosslinking capacity (->adhesive ability) depends to a large extent on its polyphenolic moieties that generate free radicals. In theory, this can lead to activated oxygen species then oxidative damage to the cell plasma membrane. I am curious if anyone has ever tested Cell Tak to evaluate damage that it might cause to cells. This could show up as an increased permeability to Ca++. There must be lots of Cell Tak experts out there. Any thoughts? Mario >Last week I had some users bring in suspension >cells for me to image with our confocal. Darn >things were moving so much that it was difficult >to get two colors to line up using sequential >scanning. Apparently these are non-adherent >cells, so growing them on a coverslip is not an >option. Any suggestions? What they gave me was >a drop of cells in culture media, we put it >between a slide and coverslip. > >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" > -- ________________________________________________________________________________ Mario M. Moronne, Ph.D. [hidden email] [hidden email] [hidden email] |
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In reply to this post by cromey
We are looking for linear polarizing filters that work in the UV range to place in the excitation path of a widefield microscope.
The standard filters used in fluorescence microscopy are limited to the visible range and the only polarizing filters operating around 360nm that we have come across seem very expensive, >700 dollars.
A promising material, HNP'B, was available from the Polaroid Corp is apparently no longer available.
any suggestions would be appreciated.
Jeremy Adler Cell Biology The Wenner-Gren Inst. Arrhenius Laboratories F5 Stockholm University Stockholm 106 91 Sweden |
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In reply to this post by cromey
I have not tried them but Nunc has a product called the Livecell Array that
has little wells that the cells sit in. If I remember they are expensive but perhaps they can be used multiple times. http://www.nuncbrand.com/en/page.aspx?ID=11388 No commercial interest. Sincerely, Claire Brown |
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In reply to this post by Mario-2
Hi,
We use 3-aminopropyltriethoxy-silane coated coverslips to study live T cells, both primary T cells and Jurkat T cells. There does not appear to be any adverse effects on the cells. Polylysine coated coverslips on the other do cause T cell spreading and activation. Regards, Ingela -----Ursprungligt meddelande----- Från: Confocal Microscopy List [mailto:[hidden email]] För Mario Skickat: den 6 oktober 2008 20:34 Till: [hidden email] Ämne: Re: mounting suspension cells Doug, You did not mention exactly what you were trying to image in/on the cells. Cells that have been developed to grow in suspension have been selected for this ability and they do not generally have the ECM/integrin/cytoskeletal network that provides surface adhesion. Then, of course, someone may have simply given you some freshly trypsinized cells resulting in the same problem, namely the lack of surface adhesion molecules. In any case, if the cells do not have to be imaged live, cells can be gently pelleted and washed a couple of times with iso-osmotic PBS to eliminate extraneous proteins from growth medium. After a couple of rinses the cells can be suspended then fixed with the usual reagents say 2% paraformaldehyde in PBS. After 30 minutes to an hour the cells will usually be sufficiently permeabilized to allow labeling with Ab fluorophore conjugates, etc. Conventional labeling on coverslips or slides is easily adapted to perform in suspension using gentle pelleting and resuspension for rinsing cells. As for mounting in something that is ~ physiological, I like to use a solution of 50% glycerol plus PBS. The glycerol viscosity helps keep cells from moving around (more glycerol helps, too), and because of the lower dielectric constant, the electrostatic binding to coverslips or slides treated with polylysine also helps with adhesion (must have M.W." 300 kDa). Even better would be to make some aminopropyl silane treated slides and coverslips. Polylysine can come off the glass and wrap cells together in the extreme. The aminopropyl silane treatment creates covalently bound positively charged surfaces that won't come off (the amino group can become oxidized but takes a very long time). Although I have not tried this with live cells, aminopropyl glass would be first thing I'd try. However, there is a potential problem with using anything that creates a very large electrostatic field in that this could have a strong effect on the PM causing channels to open/close, local lipid rearrangement (lipid flip-flop), and generally altered kinetics of membrane transporters and receptors. Cell Tak as Nathan suggested could prove advantageous for live cells; however, some of the same problems could apply in that anything you do to cells that are without the capacity to adhere are likely to experience significant perturbation in their cell surfaces. Further, I also have a concern about Cell Tak in that its crosslinking capacity (->adhesive ability) depends to a large extent on its polyphenolic moieties that generate free radicals. In theory, this can lead to activated oxygen species then oxidative damage to the cell plasma membrane. I am curious if anyone has ever tested Cell Tak to evaluate damage that it might cause to cells. This could show up as an increased permeability to Ca++. There must be lots of Cell Tak experts out there. Any thoughts? Mario >Last week I had some users bring in suspension >cells for me to image with our confocal. Darn >things were moving so much that it was difficult >to get two colors to line up using sequential >scanning. Apparently these are non-adherent >cells, so growing them on a coverslip is not an >option. Any suggestions? What they gave me was >a drop of cells in culture media, we put it >between a slide and coverslip. > >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" > -- ________________________________________________________________________________ Mario M. Moronne, Ph.D. [hidden email] [hidden email] [hidden email] |
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