I am working with mouse mutants that have an increased level of apoptosis during development, though they are otherwise indistinguishable from their het/wt litter mates. I am hoping to reduce wasted time and materials by only performing in vitro experiments on nulls and a suitable number of controls.
I would like to know if anyone has any suggestions about a rapid way to genotype/phenotype these animals based upon a viability stain, or if anyone has a rapid (
I generally use Annexin-V + 7-AAD staining to do this, and it is reasonably effective. If you don't need the cells alive, you can fix/perm and stain for cleaved caspase.
I generally use Annexin-V + 7-AAD staining to do this, and it is reasonably effective. If you don't need the cells alive, you can fix/perm and stain for cleaved caspase.
Thanks for the answers. Do you think either of these methods (pSiva/AnnevinV) would work in live Vibratome sections?
For non-fixed cells I use Cytrak Orange (goes into all cells) and DRAQ7 (outcompetes Cytrak Orange in dead cells) from Biostatus for flow cytometry viability assessments. These dyes were however fist developed for microscopy. You could contact them - they are super helpful: [email protected]
Hi Chris,
The most commonly used method to detect cell death on tissue sections is the TUNEL assay. It is used to detect apoptotic cell death but may in some instances also deteect necrotic cell death (Gavrieli et al., 1992). You can also stain your sections with an antibody that recognizes processed caspase3, but this will only detect apoptotic cells.
Using cell-impermeable dyes on sections as suggested above is a bit tricky, as cells rupture during the process of making sections, creating high background. In vivo-injection of propidium iodide has been described; this way, the cells are stained before the animal is sectioned and cells are damaged (You et al. 2008) A cell impermeable stain that recognizes the extracellular DNA released by dying cells is described in Dasari et al 2010.
We just published a book chapter on detection of cell death and distiction between apoptosis and necrosis, I can send it if you're interested (Grootjans et al, 2014).
Hope this helps,
Sasker
Gavrieli Y, Sherman Y and Ben-Sasson SA (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119:493-501.
Dasari M, Lee S, Sy J, Kim D, Brown M, Davis M and Murthy N (2010) Hoechst-IR: an imaging agent that detects necrotic tissue in vivo by binding extracellular DNA. Org Lett 12:3300-3303.
You Z, Savitz SI, Yang J, Degterev A, Yuan J, Cuny GD, Moskowitz MA and Whalen MJ (2008) Necrostatin-1 reduces histopathology and improves functional outcome after controlled cortical impact in mice. J Cereb Blood Flow Metab 28:1564-1573.)
Hi Chris,
I forgot to add that some people do PCR for presence of mitichondrial genes in the blood, released by broken mitochondria from dying cells.
good luck,
sasker
You may also try double stainings with calcein to label all cells and propidium iodide, to label dying cells (e.g. LiveDead assay from Molecular Probes). If apoptosis is of interest I would employ caspase-3 stainings with a fluorescent dye (e.g. DEVD-CHO) cleaved by caspase(s). Both assays work well in life cell imaging and should be fixable afterwards. However in thicker vibratome sections, dye penetration may really be an issue. TUNEL may be the gold standard, but its not too easy to handle. Good luck!
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