I want to test for apoptosis in endothelial and smooth muscle cells. What are some reliable methods or assay kits for testing apoptosis?
Article Apoptosis Assays
https://www.thermofisher.com/us/en/home/references/protocols/cell-and-tissue-analysis/apoptosis-protocol.html
BD Pharmingen™ FITC Annexin V Apoptosis Detection Kit, San Diego,CA, USA
https://www.citeab.com/kits/10163113-556570-bd-pharmingen-fitc-annexin-v-apoptosis-dete
Hi,
I use FITC Annexin V Apoptosis Detection Kit with Propidium Iodide from Biolegend.
Method of detection via flow cytometry.
There are many ways to investigate apoptosis in vitro.
As mentionned above, you could use any of these kits for Annexin V-PI staining. This will quantify phosphatidylserine translocation to the outer layer of cell membrane (an early marker of apoptosis).
You could also use a caspase3/7 activity probe from invitrogen. Its considered as an early marker too.
You could do cell cycle analysis by flow cytometer with PI/RNase solution to quantify DNA fragmentation. You coukd obersve DNA fragmentation by agarose gel. And finally, you could blot markers from the apoptotic pathway by Western Blot!
For flow cytometry, I use preincubation with CaCl2 2.1 mM + Alexa647 Annexin V from Biolegend.
Kits usually have the concentration of CaCl2 2.5 mM, but I haven't seen much of a difference between 2.5 and 2.1, but with 2.1 mM you have a lesser chance of additional cell activation.
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