Apoptosis detection via automated algorithms to analyze biomarker translocation in reporter cells

Abstract

Rapid, efficient, and robust quantitative analyses of dynamic apoptotic events are essential in a high‐throughput screening workflow. Currently used methods have several bottlenecks, specifically, limitations in available fluorophores for downstream assays and misinterpretation of statistical image data analysis. In this study, we developed cytochrome‐C (Cyt‐C) and caspase‐3/‐8 reporter cell lines using lung (PC9) and breast (T47D) cancer cells, and characterized them from the response to apoptotic stimuli. In these two reporter cell lines, the spatial fluorescent signal translocation patterns served as reporters of activations of apoptotic events, such as Cyt‐C release and caspase‐3/‐8 activation. We also developed a vision‐based, tunable, automated algorithm in MATLAB to implement the robust and accurate analysis of signal translocation in single or multiple cells. Construction of the reporter cell lines allows live monitoring of apoptotic events without the need for any other dyes or fixatives. Our algorithmic implementation forgoes the use of simple image statistics for more robust analytics. Our optimized algorithm can achieve a precision greater than 90% and a sensitivity higher than 85%. Combining our automated algorithm with reporter cells bearing a single‐color dye/fluorophore, we expect our approach to become an integral component in the high‐throughput drug screening workflow.

Quantitative confocal microscopy for grouping of dose–response data: Deciphering calcium sequestration and subsequent cell death in presence of excess norepinephrine

“…Although measurement of higher concentrations of Ca 2+ is one of the limitations of the proposed technique, the error arising from unwanted pixels was minimized using background correction, as mentioned in Supplementary Figure S1. Although the expression of single reporter biomolecules can be used for live monitoring and for generating dynamic data on apoptotic processes using high-resolution microscopy, 40 the major disadvantage is lower transfection efficiency. This method has the ability to monitor transfected cells, which are around 30-40% of the whole cell population.…”

“…Fluorometric characteristics have to be chosen such that there are no overlapping signals. 33,40 It was observed that imaging with multiple dyes, including PI-Fluo-4 and Hoechst-Fluo-4, leads to overlapping signals, different rates of internalization of the dye, and modification in toxicity levels. Hence, to quantify the toxicity or lytic process in the presence of norepinephrine, it was not possible to perform imaging using combinations of Fluo-4 and PI or Fluo-4 and Hoechst.…”

Quantitative Confocal Microscopy for Grouping of Dose–Response Data: Deciphering Calcium Sequestration and Subsequent Cell Death in the Presence of Excess Norepinephrine

“…Although the expression of single reporter biomolecules can be used for live monitoring and for generating dynamic data on apoptotic processes using high-resolution microscopy, 40 the major disadvantage is lower transfection efficiency. This method has the ability to monitor transfected cells, which are around 30–40% of the whole cell population.…”

“…Fluorometric characteristics have to be chosen such that there are no overlapping signals. 33,40 It was observed that imaging with multiple dyes, including PI–Fluo-4 and Hoechst–Fluo-4, leads to overlapping signals, different rates of internalization of the dye, and modification in toxicity levels. Hence, to quantify the toxicity or lytic process in the presence of norepinephrine, it was not possible to perform imaging using combinations of Fluo-4 and PI or Fluo-4 and Hoechst.…”