Abstract 609: Novel techniques for identifying apoptosis with pSIVA-IANBD in live and fixed cells.

Historically, flow cytometry has been the primary technique for identifying individual cells undergoing apoptosis. However, imaging is becoming increasingly important for analyzing cell death and underlying mechanisms in living cell populations. In this study, we developed new techniques for using the polar sensitive pSIVA-IANBD Annexin XII probe to identify apoptosis in mixed populations of cells by multiple imaging of viable primary neuronal cultures over 0-72h. The pSIVA-IANBD probe was added to living cultures at time=0. pSIVA-IAND binds to exposed phosphatidylserine (PS) residues and fluoresces (in the FITC channel) only when bound to cells. Cultures were fixed and permeabilized at the end of their respective time courses and immunostained with MAP2 or NeuN neuronal markers to identify neurons, and counterstained with Hoeschst dye to visualize nuclei. The results were imaged by an IFL multichannel inverted digital microscope (EVOS; AMG Inc, USA). They showed that bound pSIVA-IANBD was fixation resistant and the single (pSIVA-IANBD), double (pSIVA-IANBD + Hoescht) and as well as the triple (pSIVA-IANBD + Hoescht + antibody) stained cells were readily identifiable in the mixed cell population. They suggested that pSIVA-IANBD enables detection of PS, a hallmark of apoptosis by imaging of viable as well as post-fixed cells. Furthermore, since pSIVA-IANBD is not toxic to cells and does not require a washing step, it can be maintained in cultures with only a single application and no medium change for up to 72h. This allows continuous live monitoring of cells for apoptosis, cells can then be fixed and labeled with additional biomarkers of interest and imaged. These features render pSIVA-IANBD a particularly useful tool for toxicological studies of novel drugs in cell-based assays. pSIVA-IANBD is also suitable for flow cytometry and may supercede the widely used Annexin V-FITC PS probe which has has limited applications outside of flow cytometry. Citation Format: Lisa S. Heiden, Stan Krajewski, Payton D. Quintel, Sujay K. Singh. Novel techniques for identifying apoptosis with pSIVA-IANBD in live and fixed cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 609. doi:10.1158/1538-7445.AM2013-609