> We have a couple of users who want to use the confocal to look at calcium
> and/or pH changes on suspension cells (culture cells).  Does anyone know of
> specific tricks for trying to stabilize single/suspension cells in a
> perfusion chamber.  We would like to follow a single/small group of cells
> throughout treatment with different agents.  We have tried both charged and
> coated coverslips (silane and gelatin) without sucess.  Any and all help
> would be greatly appreciated.
 
We have used both embedding and coating techniques, and both are
excellent for specific applications.
 
COATING COVERSLIP:
will only be appropriate if the cells are not markedly
affected by attachment, but is probably preferable if you need
to effect rapid solution  changes and follow cell responses in the
millisecond range.
  Cell-Tak (Collaborative Biomedicals, part of  Bectin Dickinson): it's
relatively cheap and easy to prepare, use about 1ug per coverslip....
attachment is fairly rapid.
  Silanes, such as 3-aminopropyltriethoxy-silane (Sigma), are also
excellent. Excellent attachment of sections, but for (cardiac) cells
Cell-Tak is better [our experience].
 
AGAROSE EMBEDDING:
we embed sperm in 1% agarose made up in buffer.
It's a low temp gelling and avail from most suppliers. Warm to
45degr. to liquify, then bring down to 30 degree, introduce the
cells, pour (or cast a thin blanket of cell suspension in chamber) and rapidly
cool on ice for a few min.  We monitor changes in living sperm,
immobilised but otherwise fully functional at 37 degrees.
 
By casting a thin layer of the agarose suspension, you would be able to
perfuse the cells without movement, and it might be an idea to first coat the
coverslips as well. Belt and braces always keep the pants up !
 
HINTS.. for embedding technique
1. You may want to perfuse the chamber and cell preparation with a dye
to check the diffusion/perfusion. Sodium fluorescein  or any other
fluorescent solutions are OK for this.
2. Suggest you also compare loading the fluorophore before and after
embedding.
 
PS. Let us hear about your success(es).
 
Ian.
 
 
 
*********************************************
Dr Ian Harper
Experimental Biology Programme
Medical Research Council
PO Box 19070              Tel: 027-21-938 0347
Tygerberg 7505            Fax: 027-21-938 0456
South Africa
Internet: [log in to unmask]
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