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Hi Everyone, After reading the discussion here, I'm going plate some cells on 3-aminopropyltriethoxy-silane (APES) treated coverslips and compare cellular Ca++ concentrations to that in cells plated on CellTak (and, time-permitting, to that of cells on poly-L-lysine coated coverslips). I'm using two different lines of mast cells (HMC1 and LAD-2).
The protocol I'm planning to use to treat the coverslips is from Curtis and Buchan, Methods in Molecular Medicine, vol. 107: Human Cell Culture Protocols, Second Edition ( http://www.springerlink.com/content/p4v114n573p32856/fulltext.pdf). It is:
"24-well plates with APES (3-aminopropyltriethoxy-silane, Sigma) coated cover slips (12 mm) are optimal for fluorescence ICC and confocal microscopy. Prior to coating the cover slips with APES, they are first cleaned with 1% HCl/70% ethanol for 30 min, rinsed well with dH2O, soaked in 70% ethanol for 30 min, rinsed with H2O, then dried in an oven. Two percent APES in dry acetone is poured over the cover slips, and cover slips are swirled in a Petri dish for 5 s. The APES solution is poured into a waste bottle and the cover slips are washed thoroughly in dH2O, and dried in an oven. Place the coated cover slips into each well of a 24-well culture plate and expose the plates to UV light in the hood for 30 min. Store plates at 4°C until required."
Hopefully, that's reasonable. Also, how long have people let their cells attach to APES-treated coverslips? For CellTak, an hour or two results in plenty of attached cells. For poly-L-lysine (using the protocol listed earlier in this thread) I attach overnight.
Thanks so much, Nate Nathan O'Connor Silver Laboratory Department of Physiology and Biophysics Weill Cornell Medical College New York, NY 10006
From: Ingela Parmryd [mailto:[hidden email]]
Sent: Wed 08/10/2008 15:12
Subject: SV: mounting suspension cells
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 [[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]
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-- Nathan O'Connor
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