Re: Viability Staining on Frozen Sections ?

From:Greg Dobbin <dobbin@Upei.CA> (by way of histonet)

Hi Mike,

Let me first qualify my response with: I have only a rudimentary
experience with gel matrix and cell culture. I have done virus
isolation in in such systems, a few years back.

I am wondering, first, if the matrix is so viscous, how will the
nutritional requirements of the cell be replenished? And, second,
how will the metabolic wastes be effectively removed? If you' have
these questions addressed, and are satisfied there is sufficient
movement of materials, then how about this: a radio-lable on one of
the nutritional elements like an essential amino acid in the media,
then somehow seperate the cells from the media so you can
quantify uptake. This idea presumes that the cells would continue to
accumulate radiactive lable until death. Of course, I have no idea if
this is the case! (I just enjoy brain-storming).

On the other hand, as a cell dies, changes in the nucleus should be
readily evident in an H & E. You don't need TUNEL to demonstrate
pyknotic nuclei.
Again Mike, I am no expert. I'm just tossing my 2 cents worth in! Let
us know how it turns out. Cheers! Greg

Date sent:      	Tue, 12 Sep 2000 10:08:56 -0400
From:           	Michael Archambault <marchambault@osiristx.com>
Subject:        	Viability Staining on Frozen Sections ?
Forwarded to:   	DOBBIN@acad1.cs.upei.ca
To:             	'HistoNet Server' <HistoNet@pathology.swmed.edu>

> Good Morning All
>
> I was hoping to pick your brains, and see what people are using for cell
> viability stains, specifically if anything has been worked out for use on
> frozen sections.  To briefly outline what I'm looking for:  I'm going to be
> trapping cultured cells within a gelling matrix of limited diffusibility,
> with the goal of determining if cells remain viable during the in-vivo
> degredation of the gel.  The gel is quite viscous to start with, and we're
> formulating the gel to contain nutritional support, hopefully to boost the
> cell's survival rate.  In the past, I've worked with collagen gel matrices,
> gelatin, alginate, and other types of matrices, all with greater diffusion
> properties than the new matrix under investigation.  Generally I've been
> working with fluorescent nucleic acid dyes (propidium iodide, dapi, SYBR
> green, ethidium homodimer) that are excluded from live cells but are taken
> up by cells with compromised membranes.  These all work well for the
> "looser" more fluid permeable matrices which allow good dye diffusion within
> the matrix, but I'm concerned that the new matrix will not allow dye
> diffusion in a time frame that cells may be assayed.  Hence I'm trying to
> look for another method to determine the cells' viability.  Our first round
> of experiments will be in-vitro, looking at a time course of cell survival,
> in the range of 12 hours to 1 week.
>
> So, in brainstorming this new question, I was considering using some of the
> metabolic activity measures such as tetrazolium salts or dihydrofluoresceins
> to measure oxidative or reductive activities respectively, or one of the
> non-specific esterase activity dye substrates.  I was wondering if anyone
> knows if these methods may work with frozen sectioned material, or what
> other methods people may be using to address this viability question in
> tissues.  I'm not primarily interested in apoptosis (TUNEL) methods, and
> other indicators of cell death such as looking for macrophage infiltration
> since the first experimental round will be in-vitro.
>
> Any input would be most welcomed.  Thanks
>
> -Mike
>
> Michael Archambault, Research Scientist
> Bone and Soft Tissue Program
> Osiris Therapeutics, Baltimore MD
> 410 522-5005 x 226
> marchambault@osiristx.com
>


>
>
>
>
>
>
Greg Dobbin
Pathology Lab
Atlantic Veterinary College, U.P.E.I.
550 Unviversity Ave.
Charlottetown, P.E.I.
Canada,  C1A 4P3
Phone: (902)566-0744
Fax: (902)566-0851




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