I need to detect apoptosis on bovine monocyte-derived macrophages. I induced apoptosis on the cells by heating them (43°C for 3 hours), I could confirm apoptosis by morphology, however I couldn't detect any positive population to Annexin V-FITC with flow cytometry. The kit from ebiosciences said that reactivity was only for mice, rat and human, however I can't think a reason why it didn't work since most kits show reactivity to mammals. Most papers on cattle cells use TUNEL assay, but I do not have any experience with it. I'm wondering if someone could please help me with this issue. Thanks!
Since Annexin binds phosotidylserine, it in theory shouldn't matter from what species it comes from so the kits should work. When you did the assay, did you resuspend your samples in FACS buffer or did you use the Assay Buffer described in the kit? It needs to be left in a buffer that has calcium or else the binding won't work (unless you fix). FACS buffer usually has EDTA in it that will chelate the calcium.
There are some circumstances in which annexing can give misleading results (Cytometry 44:65–72 2001). TUNEL plus morphology is a really good combination. I have some nice micrographs in which the nuclear fragmentation, decreased cell size, and apoptotic bodies are clearly visible in TUNEL positive cells by fluorescence microscopy.
To quantify, I'd say Annexin V. I haven't had problems with that so far in detecting apoptosis of human monocyte derived macrophages
Hi I think the main problem in your annexin V ssay is the buffer, it did not have calcium. We have used this assay and it works quite well for cells of any species. Concerning morphology and TUNNEL recall that you should use at least 3 methods to detect apoptosis and they could be useful, otherwise use FLICA for flow cytometry or molecular biology.
good luck
Annexin V should work since it binds to phosphatidylserine at the outer leafelet of the plasma membrane. I recommend to try another probe. I use annexin-V labeled with APC (giving a strong signal) in combination with a special binding buffer, both provided from Becton Dickinson. You should also try the TUNEL assay (perhaps you can learn it in a laboratory which is familiar with it. In respect to morphology, transmission electron microscopy is the golden standard technique for detection of apoptosis. I wish you a good success!
The problem with FITC using Annexin is that phagocitosis of apoptotic cells masks membrane markers like phosphatidyl serine. Try to asses al shorter incubation times to find PS exposition before they were phagositosed. Another interesting assay is analyzing cell cicle with IP od DAPI on cells fixed with 70% EtOH. Sub G1 pic reveals cells that loose DNA content.
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