I know you can label for apoptosis markers to check if cells in tissue are dying, but what would you recommend to show that tissue is viable outside of morphology.
Fixation will punch holes in all the membranes to some extent. So any of the vital dyes used to differentiate viable from non-viable cells would give a 100% dead result. You will also hit issues of dye penetration. In my experience vital dyes will only get in a couple if cell layers in a live tissue.
Hi, Amy,
you can stain for cell vaibilty only before foxation, by using some fluoresent dyes, for example. Once you fix cells, you cant directly detremine cell viability direct, but may have indirectly determine by observed the cells structure and presence of specifci proteins, like membrane porteins. However, you will not have very precise information.
Living cells can be stained with vital stains. Intra- vital and supra vital stains are available- Toludine blue and Shillers iodine are intra- vital stains where as Brilliant crysyl blue,annexin-5,propidium iodie etc are supra vital stains. Once you fix the tissue,fixation artifacts will be formed in the tissue. While examining stained tissue or cell samples we can get an idea whether it had undergone post-mortem changes before fixation.Cytoplasmic vacuolation, pale chromatin,enlargement of nuclei etc are characteristics of these changes.It requires some experience in microscopy of cells and tissues.
Tryphan blue is also a supra vital stain which is often used to assess cell viability in culture.
Even if any of the vital stains survive fixation, you are still going to have issues will penetration of the dye. The best penetration i have acheived in live/unfixed tissue is 5-6 cell layers using Draq5, but that stains all nuclei. We have tried other dyes for necrosis, apoptois etc in the past and the best we get is around 2 cell layers. Electroporation is one way to get better penetration, buit your dye will need a charge and there will be no garuntee it will get evenly distributed. DMSO in the media is another possibiliy to get etter peneration, but its success appears to be tissue type dependant.
You can mix the cell suspension with your stain and then prepare the smear or if you want to stain smears you can try mono layer preparation using cytocentrifuge or membrane filter or even density gradient solutions.
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