Hi all!
I'm wondering if it is possible to stain my cells with annexin V before performing the surface staining. My surface staining might take up to 4 hours and I'm afraid that my cells might enter apoptosis during this time.
Any advice and suggestions woube be greatly appreciated.
Hi Mateusz,
I believe with most kits you have to keep a "binding buffer" in the cell solution along with the Annexin V. This may be cumbersome/expensive if you do it in the beginning because you would need to keep adding the binding buffer in each step of your subsequent 4 hour process to keep the Annexin V bound. I'm curious what antibodies require such a long incubation? Perhaps people can help refine your antibody labeling process to decrease the possibility of apoptosis if it's not sensitive information and you want to share it, just a thought.
Best,
Rich
Also, Purdue's flow core has a great listserv for Flow Cytometric questions. Many experts that respond with great information. It is searchable too if you notice in the right hand column of their webpage. http://www.cyto.purdue.edu/
Hi Richard!
Thanks very much for your message! Yes , I'm aware that it might be quite an expensive procedure, but it seems that I don't have another choice. My surface staining, beside direct conjugated antibodies, is going to include two different primary antibodies which then have to be detected by the secondary antibodies. I was thinking about adding direct conjugated antibodies and primary antibodies at the same time followed by adding secondary antibodies. However, I think it would be "'safer'' to add the first primary antibody followed by its secondary antibody and subsequently add the second primary antibody followed by its secondary antibody. I ran a pilot experiment a couple of days ago and the above mentioned staining took about four hours. When I run my samples on FACS, my cells (neutrophils) were all positive for Annexin V.
I guess that I’m just going to keep everything in binding buffer and hope that annexin remains bound :)
Thanks for the link to the website. It looks like it might be really helpful!
Best,
Matt
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