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 best when properly regulated—too much can result in pathologies such as Alzheimer’s, for example, while too little may result in tumor growth.
An important regulator of apoptosis is a family of 12 proteases known as caspases. Some of these, known as initiator caspases, help stimulate the cascade of signaling events that kick-starts apoptosis. Others, called effector caspases, execute the apoptotic pathways themselves.
Given their involvement in apoptosis, caspases and their enzymatic activities are often used as biomarkers for that process. Most caspase assays use fluorometric, colorimetric or flow cytometry-based methods and come in easy-to-use kits that make implementation a breeze.
Fluorometric and colorimetric caspase assays
Fluorometric and colorimetric assays use caspase enzyme activity and a specially designed reporter substrate to generate either a fluorescent or colored (or sometimes luminescient) signal, which can either be read by a microplate scanner or imaged microscopically. “A short peptide that contains a specific caspase-recognition sequence is used as a substrate and linked to a developer molecule,” says Miriam Ferrer, assays product manager at Abcam. “Upon cleavage of the substrate, the developer molecule will change color or emit fluorescence that will be directly proportional to the activity of the caspase present in the assay.”
Fluorometric assays are more sensitive—on average, 10- to 100-fold more so than colorimetric-based assays, according to Ferrer—and sometimes measure across a wider dynamic range, as well. Another advantage is that fluorometric assays “tend to have lower background, because the only thing contributing to the signal is the released fluorophore,” says Courtney Noah, senior product manager at Enzo Life Sciences. But fluorometric caspase assays also require specialized instrumentation and materials that may incur extra costs—for example, black-bottom microplates and special filters for the chosen fluorophore.
In colorimetric assays, the signal is a visible change in the reaction’s color. “This does require a low background, though, or at least a bulletproof background and negative control,” says Noah.
For purified samples, Noah says, either approach will work, but, “We tend toward colorimetric for analysis of recombinants, because the readout is easier and linked directly to a physical value.”
Colorimetric and fluorometric caspase assays are available from such vendors as Abcam, BioVision, Enzo Life Sciences, GenScript, Life Technologies, Promega (which also offers luminescence-based caspase assays), PromoKine and Sigma-Aldrich. If you aren’t sure whether you’d be better off with a fluorometric or colorimetric assay, or perhaps need both, some companies (such as Enzo Life Sciences) offer caspase assay kits that include both substrate types.
Flow cytometry caspase assays
Flow cytometry-based caspase assays provide a different kind of information than their fluorometric and colorimetric cousins. These assays measure caspase activity cell by cell as the cells pass through a flow cytometer. This approach is particularly useful if you want to measure the proportion of cells that are showing caspase activity, for example, in response to a specific experimental treatment.
Flow-based caspase assays are also helpful for studying individual cellular responses, in contrast to the average response of all cells. “An entire population will seldom respond equivalently across the population, so there is valuable additional information to be gained by observing the distribution of cells exhibiting caspase activity in a flow experiment,” says Rick Wiese, R&D manager, research content and reagents, at EMD Millipore.
Another benefit of using flow-based caspase assays is the ability to make multiple measurements per cell simultaneously—for example, by using different probes for caspase activity and cell-death markers. In this way, “researchers can distinguish early- to mid-apoptotic cells from late apoptotic/dying cells,” says Kamala Tyagarajan, senior R&D manager at EMD Millipore. “Researchers can also distinguish cells that might be exhibiting different caspases at different levels, or study caspase level changes with regard to mitochondrial potential changes using multiparametric approaches.”
On the other hand, while flow-based caspase assays can work with both suspension and adherent cell types, case must be taken with the latter as the process of preparing adherent cell types for flow cytometry sometimes can initiate early apoptotic responses, says Wiese. Another potential disadvantage, according to Camilo Moncada, staff scientist at Rockland Immunochemicals, is that “cells need to be permeabilized for detection, [so] it is important to include additional controls for other processes like necrosis, which causes the cell membrane to become permeable.”
Flow cytometry-based caspase assays include Abcam’s Generic Caspase Activity Assay Kit and Caspase 3 and Caspase 9 FITC staining kits; Enzo Life Sciences’ CaspSCREEN™ flow-cytometric apoptosis-detection kit; Life Technologies' CellEvent® Caspase-3/7 Green Flow Cytometry Assay Kit; and EMD Millipore’s FlowCellect® MitoCaspase 3/7 Assay, Muse™ Caspase-3,7 kits and Muse MultiCaspase no-wash kits for the company’s Muse Cell Analyzer.
EMD Millipore also offers an alternative method of measuring caspase activity. The company’s MILLIPLEX® MAP multiplex immunoassays include two caspases along with other apoptosis markers, which are read using Luminex microbead instruments. “By using a multiplex approach, we are able to look at multiple markers, or indicators, that a cell population is progressing down an apoptotic path,” says Wiese.
Indeed, though caspase activity is a reliable indicator of apoptosis, it is not an absolute guide. Thus, it makes sense to do the right controls, as well. “Cells will still undergo apoptosis when caspases are inhibited,” says Ferrer. “Therefore, caspase assays should always be done in conjunction with other apoptosis assays, such as phosphatidylserine exposure or chromatin condensation.”
Moncada agrees that measuring one parameter isn’t enough when studying apoptosis and advises using additional assays “to demonstrate the absence or presence of other processes like necrosis, autophagy or senescence.”
Image credit: Life Technologies. Multiplex imaging of U2OS cells using CellEvent™ Caspase-3/7 Green detection reagent.