Viability Staining on Frozen Sections ?

From:Michael Archambault <> (by way of histonet)

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


Michael Archambault, Research Scientist
Bone and Soft Tissue Program
Osiris Therapeutics, Baltimore MD
410 522-5005 x 226

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