Apoptosis Assays by Flow Cytometry

Apoptosis Assays by Flow Cytometry

Flow cytometry is a flexible and versatile method for measuring populations of cells undergoing apoptosis, or cell death, at different stages. Early in apoptosis, the mitochondria lose membrane potential and are unable to function properly, leading to cytochrome c release and the activation of caspases. Flow cytometry enables the detection of activated caspases in cells with the use of probe molecules containing a cleavage site specific for caspases 3 and 7.  When cleaved by these activated caspases in early apoptotic cells, the probe molecules enter the nucleus, bind to DNA, and fluoresce.

The early stage of apoptosis also involves phosphatidylserine (PS) translocation from the inner cellular membrane to the outer leaflet, exposing it to the extracellular environment.  During later stages of apoptosis, cellular membranes become permeable, compromising their integrity and selective transport of materials. Flow cytometry reagents such as annexin V can be used to bind exposed PS on cell membranes for labeling of early apoptotic cells, while the DNA binding dye 7-AAD can be used to label late apoptotic cells.

High-performance flow cytometers often require training or an extensive amount of experience to deliver high-quality results. Designed to meet the needs of the most demanding scientists while still being accessible for novice users, Agilent NovoCyte flow cytometers make it easy to preform apoptotic assays. Discover a line of flow cytometers that provides:

• High-quality scatter resolution for small particle detection

• Consistent results at varying flow rates

• Accurate absolute cell counts, rendering reference beads unnecessary

• Easy maintenance, automatic cleaning cycles

A Powerful Yet Affordable Flow Cytometer for Apoptosis Assays

NovoCyte flow cytometers are built with trusted Agilent quality and have a proven track record of delivering reliable performance. Avoid the hassle of manual maintenance or the need to routinely adjust your detector settings. An advanced fluidic design gives you the reproducibility you require, while also offering scheduled and automatic startup and shutdown, automatic cleaning cycles, and batch analysis and reporting. With a fast and robust autosampler, your flow cytometer can do the work for you and collect samples after you leave the lab. 

Detect Early and Late Apoptotic Cells using Annexin V and 7-AAD by Flow Cytometry

This apoptosis assay shows that staurosporine induces apoptosis in both Jurkat T cells and HeLa cells, providing insight into the early and late stages of apoptosis. After staurosporine treatment for 4 hours, cells were analyzed for early and late apoptotic cells by staining for phosphatidylserine with annexin V-FITC and with the DNA binding dye 7-AAD. Cells were classified as early apoptotic with annexin V-only positive staining, and late apoptotic (or dead) cells with annexin V and 7-AAD positive staining.

Measure Mitochondrial Membrane Potential Loss in Early Apoptotic Cells

Jurkat T cells are treated with staurosporine in this flow cytometric apoptosis assay, followed by the addition of the fluorescent dye JC-1. JC-1 is cell-permeable and aggregates inside mitochondria while membrane potential is maintained, emitting fluorescence in the PE channel. When the membrane potential is lost, JC-1 will not localize to mitochondria and will reside in the cytoplasm in its monomeric form, emitting fluorescence in the FITC channel. The ratio of PE to FITC fluorescence represents changes in the mitochondria membrane potential.

Using Flow Cytometry to Quantify Caspase 3 and 7 Activity in Early Apoptotic Cells

This apoptosis assay shows staurosporine-treated Jurkat T cells exhibit more caspase activity than the untreated, control cells. Jurkat T cells were treated with 2 µM staurosporine for less than 1 hour or 4 hours and stained with cell‑permeable, DEVD‑conjugated fluorescent nucleic acid binding dye and 7-AAD dye. The untreated control cells show minimal caspase activity, while cells treated with staurosporine exhibit a significant increase in caspase 3 and 7 activity (99.7%). Apoptosis Assays by Flow Cytometry

Cellular Impedance

Cellular Impedance Explained

Positioned between reductionistic biochemical assays and whole organism in vivo experimentation, cell-based assays serve as an indispensable tool for basic and applied biological research. However, the utility of many cell-based assays is diminished by: (1) the need to use labels, (2) incompatibility with continuous monitoring (i.e. only end point data is produced), (3) incompatibility with orthogonal assays, and (4) the inability to provide an objective/quantitative readout. Each of these shortcomings is, however, overcome by the non-invasive, label-free, and real-time cellular impedance assay.

Functional Unit of Cellular Impedance Assay

The functional unit of a cellular impedance assay is a set of gold microelectrodes fused to the bottom surface of a microtiter plate well (Figure 1). When submersed in an electrically conductive solution (such as buffer or standard tissue culture medium), the application of an electric potential across these electrodes causes electrons to exit the negative terminal, pass through bulk solution, and then deposit onto the positive terminal to complete the circuit. Because this phenomenon is dependent upon the electrodes interacting with bulk solution, the presence of adherent cells at the electrode-solution interface impedes electron flow. The magnitude of this impedance is dependent on the number of cells, the size and shape of the cells, and the cell-substrate attachment quality. Importantly, neither the gold microelectrode surfaces nor the applied electric potential (22 mV) have an effect on cell health or behavior.

Impedance Electrodes

The gold microelectrode biosensors in each well of Agient’s electronic microtiter plates (E-Plates) cover 70-80% of the surface area (depending if a view area is present). Rather than the simplified electrode pair depicted in Figure 1, the electrodes in each well of an E-Plate are linked into “strands” that form an interdigitating array (Figure 2). This arrangement enables populations of cells to be monitored simultaneously and thereby provides exquisite sensitivity to: the number of cells attached to the plate, the size/morphology of the cells, and the cell-substrate attachment quality.

Figure Left: Impedance electrodes on Agilent’s E-Plates. (A) Simplified schematic of the interdigitated electrodes used in each well of an E-Plate. Electrodes are not drawn to scale (only a few are shown, and they have been enlarged for clarity). Though cells can also be visualized on the gold electrode surfaces, the electrode-free region in the middle of the well facilitates microscopic imaging (brightfield, fluorescence, etc.). (B) Photograph of a single well in a 96-well E-Plate. (C) Zoomed in brightfield image of shadowed electrodes and unstained human cells. (D) Gold electrodes and crystal violet stained human cells, as viewed in a compound microscope.

Real-Time Impedance Traces Explained

The impedance of electron flow caused by adherent cells is reported using a unitless parameter called Cell Index (CI), where CI = (impedance at time point n – impedance in the absence of cells)/nominal impedance value. Figure 3 provides a generic example of a real-time impedance trace throughout the course of setting up and running an apoptosis experiment. For the first few hours after cells have been added to a well there is a rapid increase in impedance. This is caused by cells falling out of suspension, depositing onto the electrodes, and forming focal adhesions. If the initial number of added cells is low and there is empty space on the well bottom cells will proliferate, causing a gradual yet steady increase in CI. When cells reach confluence the CI value plateaus, reflecting the fact that the electrode surface area that is accessible to bulk media is no longer changing. The addition of an apoptosis inducer at this point causes a decrease in CI back down to zero. This is the result of cells rounding and then detaching from the well bottom. While this generic example involves drug addition when cells are confluent, impedance-based assays are extremely flexible and can also evaluate the rate and extent of initial cell adhesion to the electrodes, or the rate and extent of cell proliferation.

Figure Right: Generic real-time impedance trace for setting up and running an apoptosis assay. Each phase of the impedance trace, and the cellular behavior it arises from, is explained in the text.

Correlating Impedance with Cellular Phenomena

RTCA provides a quantitative readout of cell number, proliferation rate, cell size/shape, and cell-substrate attachment quality. Because these physical properties are the product of thousands of different genes/proteins, RTCA can provide an extremely wide field of view on cell health and behavior. Everything from endothelial barrier function and chemotaxis to filopodia dynamics and immune cell-mediated cytolysis have successfully been analyzed on xCELLigence instruments. Despite the breadth of their reach, xCELLigence assays are still capable of interrogating very specific biochemical and cellular phenomena. Appropriate use of controls and/or orthogonal techniques make it possible to correlate the features of an impedance trace with specific cellular/molecular phenomena. To learn more about how this is done, and to witness the sensitivity and versatility of the xCELLigence RTCA technology, peruse the many specific applications that are highlighted here.

Figure Left: Examples of real-time impedance traces obtained using E-Plates and xCELLigence RTCA instruments. (A) Real-time monitoring of A549 cell adhesion to E-Plate wells that had been pre-coated with different concentrations of collagen IV. Note the correlation between impedance values (Cell Index) and the number of adherent cells visible in the microscope. (B) Real-time impedance traces for HeLa cells exposed to different concentrations of the GPCR agonist dopamine. The black arrow indicates the time of dopamine addition. (C) Real-time impedance traces for NK 92 cell-mediated cytolysis of MCF7 breast cancer cells. (D) Real-time impedance traces for A549 cells exposed to drugs displaying a variety of mechanisms of action.

xCELLigence RTCA eSight - Imaging & Impedance

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Real-Time Apoptosis Assays

Apoptosis is a programmed and regulated process of cell death where cells activate specific intrinsic pathways that cause them to shrink, condense, and eventually be cleared by phagocytosis. Relative to necrotic cell death where cell membrane integrity is compromised, leading to cell injury and inflammation, apoptotic cell death is less destructive and limits disruption of nearby cells and tissue. Typical biomarker-based assays require multiple handling steps and only yield endpoint measurements.

Explore Agilent xCELLigence real-time cell analysis technologies to learn about a more comprehensive apoptosis assay that provides:

• Direct measurement of cell death and behavior

• Quantitative and reproducible apoptotic data

• A simple and high-throughput method

• Detailed insight into cellular mechanism of action

Multiple Perspectives into Apoptosis to Increase Confidence

From the exact same population of cells, gain deeper insights into drug-mediated apoptosis with xCELLigence RTCA eSight.  Automatically quantify and visualize apoptosis over the course of multiple days, without additional sample processing steps. Capture early and late apoptotic events such as modulation of cell adhesion, cell membrane integrity, and cell detachment. Simultaneous real-time cellular impedance and live cell imaging provide sensitive information about cell viability, growth, and morphology.

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Multiplex Impedance-Based Data with Live Cell Imaging

While the standard xCELLigence RTCA instruments (SP, MP, HT models) provide comprehensive, noninvasive and label-free analysis in 96- or 384-well formats, the xCELLigence RTCA eSight adds live cell imaging for increased confidence in your cell analysis and conclusions. In one instrument, capture five different perspectives (impedance, brightfield, red, green, and blue fluorescence imaging) on the exact same population of cells to reveal greater insight into cell health.

Visualize and Quantify Apoptosis with Agilent eSight Reagents

With three distinct fluorescence channels and brightfield imaging, xCELLigence RTCA eSight provides provides both qualitative and quantitative assessment of cell morphology, size, and confluence under various assay conditions.Combine fluorescent live-imaging reagents and dyes, such as Agilent eAnnexin V, eCaspase-3, and eLenti, for multiplexed measurements of cell viability, apoptosis, and cytotoxicity in a single well. Additionally, genetically encoded, pathway-specific fluorescent reporters are also compatible.

Determine Effective Drug Concentrations from Multiple Readouts to Confirm Accuracy

Generate dose-response curves from impedance and image-based readouts to verify the concentration of a drug that gives half-maximal response (EC50). In this example, the area under the curve, spanning from the time of drug addition to 60 hours after drug addition, can be plotted as a function of drug concentration to yield the dose-response curves. The four different readouts resulted in R2 values ranging from 0.96 to 0.98 and EC50 values from 0.86 to 3.0 µm.

xCELLigence RTCA eSight - Imaging & Impedance