Hi Silvana, Out of curiosity, what cells where you using as your positive controls, and what did you use to induce apoptosis in them? I use the AnnexinV from Pharmingen and their buffer uses 2.5mM CaCl2, and that works well for me. My positive controls are Jurkat cells with anti-CD95 and I get a good positive after 8 hours. Let me know what you use and anything else you use in your buffer. Also you may want to, if you have not already use propidium iodide to just see if you have any death occuring, just as a second control to see if you even have any dead cells. Jason Cohn Princess Margaret Hospital Ontario Cancer Institute Toronto, Canada On Wed, 3 Mar 1999, silvana wrote: > > Hi, we have problem in labeling cells with Annexin V. In our protocol the > cells are resuspended in an incubation buffer which contains 1.5mM CaCl2, > then we add 5 ul of annexin V and we leave on ice for 15'. > With this protocol we did not observed any positive cell even in the > positive control. Could anybody give us suggestions? > thank you > Silvana > >
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