pSIVA is a newly developed Annexin B12 based polarity sensitive probe for monitoring exposed PS, a hallmark of apoptosis. A unique feature of pSIVA is that it fluoresces (green channel) only when bound to PS, and hence no wash steps are needed to remove unbound probe. This attribute is enabling the development of new methodologies not previously possible with classical Annexin V conjugates, which fluoresce irrespective of whether or not they are bound to PS. In this study we developed a flow cytometry protocol whereby pSIVA is added directly to cell culture media, avoiding the use of buffers. The protocol eliminates unforeseen perturbation or negative effects that standard buffers can have on sensitive cell types. Multicolor methodology that employed pSIVA-labeled cells along with intracellular markers was developed for flow cytometry and microscopy platforms. A model using primary mouse neurons labeled with pSIVA, DAPI and MAP2 (neuronal marker) indicated pSIVA stained the cell body of spontaneously dying neurons but stained both the cell body and degenerating axons in staurosporine treated neurons. These results suggest that mechanisms of PS exposure may vary between different apoptotic processes, including spontaneous versus induced apoptosis. They also suggest pSIVA has potential for elucidating new pathways of PS exposure heretofore not possible with existing reagents.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 237. doi:1538-7445.AM2012-237