I am trying to determine whether microglia are undergoing apoptosis in an experiment. I tried Active Caspase 3 was unable to get the antibody to work (IF).
I was considering a different apoptotic marker (Annexin V), however I am unsure whether microglia express/transport phosphatidylserine to the cell surface when undergoing cell death like neurons, and whether this antibody would work.
Any ideas?
Hi Kristie,
If you have access to a fluorescence plate reader, I suggest using the AnaSpec SensoLyte Homogeneous AMC Caspase-3/7 assay kit. It was very simple and straight forward and provided pretty good results.
https://www.anaspec.com/products/product.asp?id=32762
Best of luck.
Little bit unsure about the type of microglial cell ur are handling. If you are using mouse microglial cell (BV-2) then, Annexin-V-FITC should work properly. Before doing that you can check Mitochondrial membrane potential through JC-1 in flowcytometric analysis to confirm the initiation of apoptosis. best of luck.....
It will be tricky, try to check Lindsay's suggestion. Direct and reliable
Hi Kristie, your question will be solved well when applying flow cytometry. As I used FITC Annexin V Apoptosis Detection Kit to detect cell apoptosis, this kit fit for mammal cells and plant cells, so you can have a try. And for detecting active Caspase 3, you can also use FITC( or AF647 or other fluorescence labeled) Rabbit Anti- Active Caspase-3 from BD bioscience. The two detecting method need cytometry skills and have higher sensitivity than IF or other microscope scanning ways. Good luck.
http://www.bdbiosciences.com/us/solrSearch?text=caspase3&x=0&y=0
Dear Kristie L Smith
I highly recommend using immunoblotting techniques and apply Caspase 3 antibody from Santa Cruz Biotechnology, and use it as an apoptotic marker.
Best
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