A high-throughput direct fluorescence resonance energy transfer-based assay for analyzing apoptotic proteases using flow cytometry and fluorescence lifetime measurements

Abstract

Cytometry is a versatile and powerful method applicable to different fields, particularly pharmacology and biomedical studies. Based on the data obtained, cytometric studies are classified into high-throughput (HTP) or high-content screening (HCS) groups. However, assays combining the advantages of both are required to facilitate research. In this study, we developed a high-throughput system to profile cellular populations in terms of time- or dose-dependent responses to apoptotic stimulations because apoptotic inducers are potent anticancer drugs. We previously established assay systems involving protease to monitor live cells for apoptosis using tunable fluorescence resonance energy transfer (FRET)-based bioprobes. These assays can be used for microscopic analyses or fluorescence-activated cell sorting. In this study, we developed FRET-based bioprobes to detect the activity of the apoptotic markers caspase-3 and caspase-9 via changes in bioprobe fluorescence lifetimes using a flow cytometer for direct estimation of FRET efficiencies. Different patterns of changes in the fluorescence lifetimes of these markers during apoptosis were observed, indicating a relationship between discrete steps in the apoptosis process. The findings demonstrate the feasibility of evaluating collective cellular dynamics during apoptosis.

Evaluation of Caspase‐3 Activity During Apoptosis with Fluorescence Lifetime‐Based Cytometry Measurements and Phasor Analyses

“…In order to establish a quantitative approach for measuring caspase activity, a variety of FRET bioprobes were developed and evaluated for functionality (27)(28)(29). The purpose of these BPs was to evaluate enzyme activation and identify caspase activity with precision owing to the specificity of the engineered cleavage peptide sequence (27)(28)(29). We have selected for this study an optimized and reproducible BP in order to evaluate our ability to measure the changes in the donor lifetime using time-resolved flow cytometry.…”

“…We have begun to address these challenges by utilizing Förster resonance energy transfer (FRET)-based bioprobes (BPs) combined with high-throughput screening approaches (27)(28)(29). With FRET-based BPs, developed by Suzuki et al, comprehensive caspase activity can be intracellularly evaluated.…”

Spectral Unmixing Plate Reader: High-Throughput, High-Precision FRET Assays in Living Cells

“…Conventional microplate readers, which measure fluorescence intensity, lack the precision required for reliable observation of the typically small FRET changes associated with allosteric effectors in live-cell assays. Similarly, the throughput of related fluorescence technologies such as microscopy is too low, and the precision of flow cytometry is currently too low for large-scale library screening 8,9 . The technological advances of direct waveform recording (DWR) led to the development of a novel fluorescence lifetime plate reader in the time domain 5 and now have been applied to the wavelength-domain.…”

High Throughput Screening Technologies in Biomass Characterization

“…HTP screening methods are being increasingly applied to process development in biotechnology (Long et al, 2014;Back et al, 2016;Yang et al, 2017;Zutz et al, 2017). As new methodology is developed, HTP screening is increasingly employed to collect biological data that historically required extensive time and effort (Scheel and Lutke-Eversloh, 2013;Suzuki et al, 2015) and is widely used today in the development of fermentation process assays (Decker et al, 2003(Decker et al, , 2009Selig et al, 2010Selig et al, , 2011Studer et al, 2010;Suzuki et al, 2015;Yang et al, 2017). There have been rapid developments of HTP techniques in recent years in micro-scale culturing, online analysis and monitoring, and real-time control, which have enabled increased systems automation (Yang et al, 2017).…”

“…As an example, microtiter plates (MTP) are simple, easy to shake, and inexpensive (Bharadwaj et al, 2011;Yang et al, 2017) and have been demonstrated to effectively replace shake flasks (Oguntimein et al, 2018). MTPs have been used to screen specific activities of enzyme variants using various techniques, such as protein quantitation by immunoturbidimetric (ITA) assays, (Yang et al, 2017), direct fluorescence resonance energy transfer for protease activity (Suzuki et al, 2015), cell free protein production (Casteleijn et al, 2013), fungal biosensor assay to detect estrogen activity (Zutz et al, 2017), protein purification and characterization for crystallographic studies (Kim et al, 2011), and enzyme-screening of ionic liquid pretreated lignocellulose (Bharadwaj et al, 2011). Despite these recent examples of MTP-based screening, few details are known regarding actual culture conditions inside the MTP, the technology to measure these details in real time in such numbers and small volumes remains lacking (Long et al, 2014).…”