Abstract 718: A laboratory friendly visualization method of triple-co-fluorescence staining to identify cytomorphological features of apoptosis simultaneously with biochemical events in live cells
Authors: Leyland-Jones Brian R. , Aske Jennifer H. , De Pradip , Dey Nandini 
Published: August 2019

Abstract
Background: During apoptosis, its cytomorphological features are functionally coupled to its tightly regulated and ATP-dependent biochemical events. Traditionally, identification and quantification of apoptosis in the laboratory are based on one of these features at a time from either lysed or fixed cells.
Aim: We present a protocol for staining live cells in a culture undergoing apoptosis in real-time. Live cell-imaging by Triple-co-Fluorescent Staining (TFS) is standardized to test 3 crucial events of apoptosis simultaneously in a single cell; the enzymatic activity of executioner caspase3, caspase-dependent phosphatidylserine presentation on cell-surface and mitochondrial depolarization.
Methods: Live cells were stained simultaneously with the NucView488-Casp3 substrate (G), CF594 AnnexinV (R), and MitoViewBlue (B). Specificity of the staining was tested by co-localization & caspase3 inhibitor assay. We used (1) real-time confluency (Incucyte), (2) Annexin V (Accuri C6), (3) WB expression of cl-caspase3, cl-PARP, and BIM, and (4) TMRE-A based mitochondrial depolarization on validating our method. CalceinAM green and EthD-1red were used as internal staining controls. This method was verified to test combinations of chemotherapy and targeted therapy drugs in tumor cells.
Results: Drug combinations blocked cytoplasmic B staining while increased both G & R staining compared to controls. Merged images showed 100% mutual exclusivity between B & G stains in control and treated cells as determined by overlap and co-localization coefficients. Pixel co-localization between FITC (G and CalceinAM), DAPI (B) and TRITC (R and EthD-1) channels from 2.5 nM paclitaxel + 500 nM BKM120 or paclitaxel only treated OVK18 cells confirmed above results. Caspase3 and AnnexinV staining in treated cells, however, were both separate and overlapped (yellow fluorescence) indicating the sequence of apoptotic-events. Co-localization between FITC, DAPI, and TRITC channels was determined in the scatterplot of merged channels. No co-localization between EthD-1 red and CalceinAM green cells was observed. Ac-DEVD-CHO, a tetrapeptide (amino acid sequence of PARP cleavage site) that acts as a competitive inhibitor for caspase-3/7 was used to test the specificity of the assay.
Significance: The strength of our method lies in our (a) choice of TFS, (b) independent validations, and (c) specificity of staining. To the best our knowledge this is the most stringent method to visualize 3 critical features of apoptosis simultaneously and conclusively deciphering mechanistic involvement of different stages/features of apoptosis in live tumor cells in real-time which can be applied in live tumor cells to test chemotherapy and targeted drugs’ efficacy in everyday laboratory practice with the help of a minimalistic laboratory setup.
Citation Format: Brian R. Leyland-Jones, Jennifer H. Aske, Pradip De, Nandini Dey. A laboratory friendly visualization method of triple-co-fluorescence staining to identify cytomorphological features of apoptosis simultaneously with biochemical events in live cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 718.