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The end of life, live

20th June 2018


Real-Time-Glo Annexin V Apoptosis Assay
The Clariostar plate reader with ACU

Andrea Krumm explains how to monitor cell death in real-time

Cellular microplate assays are popular tools in life science. They enable studying cellular events that cannot be analysed in isolated cell components. Monitoring such a cellular process over time expands the output of an experiment as it allows comparing timing and extent of a biological reaction and as it prevents to miss the time point of reaction. The microplate format is advantageous since various conditions can be analysed in parallel.

Recording cellular reactions in real-time requires specific characteristics of assays as well as of the measurement equipment. Obviously, a suitable microplate reader needs to be able to read the detection mode determined by the assay. For cellular assays these are predominantly fluorescence intensity, luminescence, absorbance, time-resolved fluorescence or combinations thereof. The opportunity to measure from either top or bottom of the microplate is desired to measure adherent cells and may circumvent disturbances of the measurement by cell culture medium components or condensation. An absolute requirement is the possibility to control atmosphere and temperature within the measurement chamber of the microplate reader.

To keep human cell lines viable for the duration of the experiment, they need to be incubated at 37°C and at a CO2 concentration of 5% to maintain the physiologic pH provided by a bicarbonate buffer. Since the oxygen available in human tissues is typically below ambient, it is further recommended to run real-time cellular assays at defined oxygen conditions and thereby mimic physiologic conditions.

The Clariostar multimode plate reader with atmospheric control unit (ACU) was designed to serve as an ideal platform for real-time cell based assays. Hence, it regulates temperature and atmosphere according to researcher’s needs and adds temperature and gas control data to the measurement results. The choice between top or bottom measurement is done by a simple click in the plate reader’s software.

Assay components must not be cytotoxic and should not interfere with the analysed process itself. The method must be stable for the time of analysis and at temperatures of 37°C, the temperature human and other mammalian cells are mostly cultured at. If the cellular reaction is reported by a change in fluorescence, the fluorophore needs to be resistant to bleaching and ideally emits light rather in the red range of the visible spectrum in order to be distinguishable from cellular autofluorescence.

An example for a cellular assay measured in real-time is the monitoring of two types of cell death: apoptosis and necrosis. Although cell death is an important, normal consequence of multicellular homeostasis it also has an unfortunate role in many human diseases. Discovery of targeted cytotoxic and cytoprotective agents may therefore have important implications in treating cancer, neurodegeneration and a litany of other diseases. To fully appreciate the dysregulation in a disease model, and responses to experimental stimuli, it is important establish the mechanism of action of cell death and define response kinetics. Traditional experimental approaches for addressing mode of cell death are often time consuming, labour-intensive, costly and inadequate.

A novel assay (RealTime-Glo Annexin V Apoptosis and Necrosis Assay, Promega) provides the simplicity of an add-mix-measure assay, in a live-cell, real-time format. The Clariostar with ACU provides the sensitive detection of duplexed luminescent and fluorescent signals, all while maintaining the appropriate [O2] and [CO2] environment for the cells. The combination allows walking away once the test is set up and ensures to record important cellular responses regardless of when changes happen.

The assay measures translocation of phosphatidylserine (PS) from the inner to outer membrane leaflet that is a hallmark of healthy cells transitioning to apoptosis. It uses annexin-V-fusion proteins that contain binary subunits of a luminescent enzyme (NanoBit), which are drawn into complementing proximity only due to their affinity for PS. In the presence of a time-released substrate, the complemented enzymes report real-time PS exposure. The Necrosis Detection Reagent reports changes in membrane integrity as a result of necrosis. Together, these real-time measures establish the mechanism of action (apoptosis, primary necrosis, or alternative programs) for cell death.

A dose response experiment was done with the proteasome inhibitor bortezomib and the leukemic cell line K562. Measurement of luminescence and fluorescence was done by the Clariostar every 15 minutes for 48 hours. The experiment revealed the exposure of phosphatidylserine (luminescence) before the necrotic response (fluorescence) indicating the expected apoptotic mechanism for this therapeutic proteasome inhibitor.  

The results show a combination of a novel assay methodology and instrumentation that allows the user to walk away and return to outstanding results. The real-time nature of the results allows capturing information that would require extensive effort to be achieved using previous apoptosis assay techniques.

Andrea Krumm is with BMG Labtech

 





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