Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
Apoptosis, or programmed cell death, is a complex biochemical process involving a host of different molecules, signals and pathways. One key indicator of apoptosis is activation of the caspases, a family of proteinases.
Researchers have several options for measuring caspase activity, including at high throughput. High-throughput tools for caspase assays are in demand by researchers of apoptosis-related diseases such as cancer, Parkinson’s and autoimmune diseases, as well as by drug developers. Here's what's available.
Fluorescent and colorimetric assays
One of the most common caspase assays relies on fluorescent or colorimetric readouts of caspase activity. Many are designed for a microplate-based format, which—when coupled with automated plate-handling robots—makes for powerful high-throughput capabilities. Roche Diagnostics’ Homogeneous Caspases Assay, for instance, is fluorometric and automation-friendly, and it probes caspases 3/7, 6, 8 and 10 simultaneously. “As such, it can serve as a screening tool for general apoptosis determination,” says David Guffey, senior technical services consultant in life science at Roche Diagnostics.
Key to the assay is a universal caspase substrate called DEVD-R110, a derivative of rhodamine 110. Upon cleavage, the substrate fluoresces with intensity proportional to the concentration of activated caspases. The assay requires 96- or 384-well plates and a fluorescence microplate reader, and can screen hundreds of potential apoptosis-causing compounds, which is the goal in high-throughput assays, says Guffey. But “determining additional information, such as which specific mechanisms are causing apoptosis, would require a lower-throughput assay.”
Enzo Life Sciences also offers fluorescence-based caspase assays, as well as colorimetric variants of its Drug Discovery Caspase Screening assays. “The advantage of using this type of assay is its ability to test large numbers of compounds quickly and efficiently, [which] may provide [users] a competitive advantage,” says Enzo senior marketing manager Courtney Noah. Colorimetric assays require a plate reader with spectrophotometric capability.
Flow cytometry assays
Although fluorimetric and colorimetric assays in microplate formats offer high throughput, their results reflect the average response of the cells in each well. If you need to know what’s going on in each cell of the population, consider flow cytometry.
Flow cytometry measures the fluorescence response from individual cells rather than the averaged response of every cell in the well. “Information is obtained on the distribution of cells showing caspase activity and can provide what percentage of cells exhibit caspase activity and are impacted by the treatment or condition,” says Kamala Tyagarajan, senior R&D manager at EMD Millipore.
EMD Millipore offers flow-cytometry caspase assays in both microplate and single-tube formats. The assays work especially well with EMD Millipore’s guava easyCyte™ cytometry platform, but any flow-cytometry system can be used.
The company’s inhibitor-based guava caspase kits (for caspases 3/7, 8, 9 and multicaspase) are live-cell assays that use a fluorescently labeled peptide inhibitor of particular caspases. The inhibitor enters the cells and binds to active caspases, which are then detected by flow cytometry. But they don’t measure lone caspase activity; instead, they multiplex the detection of two caspases or a caspase with another apoptosis marker. “This results in more enriched information for our researchers and provides more reliable comparative data from the same cellular sample,” says Tyagarajan.
EMD Millipore also offers more specialized assays, such as the FlowCellect™ MitoCaspase 3/7 Assay, which detects activity of caspase 3/7 along with changes in mitochondrial potential, and the FlowCellect® T-cell Caspase kits, which track caspase activity in CD4 and CD8 T cells. “These assays are highly valuable in understanding mechanism and sequence of events in the apoptotic cascade and understanding caspase-dependent and caspase-independent modes of cell death,” says Tyagarajan.
An alternative for researchers working with lysed cell samples is EMD Millipore’s MILLIPLEX® MAP bead-based multiplex immunoassays. These assays use the Luminex® xMAP® technology in kits that detect early and late apoptosis using caspases 3, 8 and 9, along with other apoptosis biomarkers. EMD Millipore also sells fluorimetric and colorimetric microplate-based assays for caspases 1, 2, 3/7, 5, 6, 8 and 9.
Luminescent assays
Another option for detecting caspase activity is luminescence. “The advantage of using luminescence is the sensitivity of detection, which is typically 50 [to] 100-fold better than fluorescent assays,” says Terry Riss, senior product specialist in cell health at Promega. That increased sensitivity "enables miniaturization of cell-based assays to 1,536-well format for high-throughput screening applications.”
Promega’s assays (for caspases 2, 3/7, 6, 8 and 9) use a four-residue peptide substrate that is linked to amino-luciferin by a peptide bond. When a caspase cleaves the peptide substrate, amino-luciferin is released and subsequently available to the light-generating enzyme luciferase. The assay kits include a detergent for cell lysis, ATP, the luciferin-conjugated peptide substrate and Ultra-Glo™ luciferase.
From a high-throughput screening standpoint, luciferase assays are advantageous because the protocol involves a single reagent addition with no washing or media removal. Throughput also exceeds typical flow-cytometry assays, Riss says, because flow cytometry “typically require[s] 10 minutes to read a plate with a single fluorescent parameter and thus require[s] staggering the production of assay plates and addition of test treatments for large screens.” There is a tradeoff, though: Luminescent assays do not measure signals per cell, as in flow cytometry, but rather signals per well.
Riss says the main application for Promega’s luminescent caspase assays is “to distinguish extrinsic (death-receptor-associated) vs. intrinsic (mitochondrial-associated) signaling pathways initiating events leading to apoptosis,” by measuring the activity of caspase-3/7 and the upstream caspases 8 and 9.
But caspase assays often serve better as markers of apoptotic phase than as a general marker of apoptosis itself. Noah says measuring a total apoptotic response would be accomplished more efficiently using a labeled antibody than enzymatic activity, for instance, as the latter can change.
“If, for instance, a caspase were highly up-regulated or activated, but then modulated down by an anti-apoptotic signal, you might be able to observe the jump in caspase activity but not an increase in apoptosis,” she says.
As researchers’ apoptotic toolbox becomes more finely tuned, they have the luxury of asking more tightly focused questions and probing ever more deeply the events that so frequently lead to cell death.