Annexin V stain control also, Why? - (Sep/25/2008 )
Hi all,
I've been struggling with detecting apoptosis in osteosarcoma cultured on my solid substrate using annexin V-FITC (Clontech). The control always show positive annexin V staining, which is not right.
This is what I do: culture the cells (adherent on my solid substrate which I believe induce apoptosis); after 12hrs, I stain both treated cells and control (which is osteosarcoma grown on glass slide) with Annexin V-FITC according to the protocol provided by Clontech. I do not detach the cells, I leave them adhered on the substrates and glass slide. Then go to florescence microscope using FITC filter.
Anyone has the same problem with Annexin V? I've heard some issues with this stain. Any ideas would be appreciated.
Thanks,
Peter
-ptives-
QUOTE (ptives @ Sep 26 2008, 04:21 AM)
Hi all,
I've been struggling with detecting apoptosis in osteosarcoma cultured on my solid substrate using annexin V-FITC (Clontech). The control always show positive annexin V staining, which is not right.
This is what I do: culture the cells (adherent on my solid substrate which I believe induce apoptosis); after 12hrs, I stain both treated cells and control (which is osteosarcoma grown on glass slide) with Annexin V-FITC according to the protocol provided by Clontech. I do not detach the cells, I leave them adhered on the substrates and glass slide. Then go to florescence microscope using FITC filter.
Anyone has the same problem with Annexin V? I've heard some issues with this stain. Any ideas would be appreciated.
Thanks,
Peter
I've been struggling with detecting apoptosis in osteosarcoma cultured on my solid substrate using annexin V-FITC (Clontech). The control always show positive annexin V staining, which is not right.
This is what I do: culture the cells (adherent on my solid substrate which I believe induce apoptosis); after 12hrs, I stain both treated cells and control (which is osteosarcoma grown on glass slide) with Annexin V-FITC according to the protocol provided by Clontech. I do not detach the cells, I leave them adhered on the substrates and glass slide. Then go to florescence microscope using FITC filter.
Anyone has the same problem with Annexin V? I've heard some issues with this stain. Any ideas would be appreciated.
Thanks,
Peter
I also used Annexin V-FITC, but I never had problems. What happens if you use only the substrate or the glass slides without cells on it, maybe there is some aspecific binding? Or else, try a caspase-glow assay or something similar, maybe you do have (more) apoptosis in your control.
Do you also use propidium iodide, because, if you observe a lot of dead cells with your substrate, it might also be necrosis.
-aspergillie-