Cell viability, cell proliferation and many important live-cell functions—including apoptosis, cell adhesion, chemotaxis, multidrug resistance, endocytosis, secretion and signal transduction—can be stimulated or monitored with various chemical and biological reagents. Many of these processes lead to changes in intracellular radicals (Probes for Reactive Oxygen Species, Including Nitric Oxide—Chapter 18), free-ion concentrations (Indicators for Ca2+, Mg2+, Zn2+ and Other Metal Ions—Chapter 19, pH Indicators—Chapter 20, Indicators for Na+, K+, Cl– and Miscellaneous Ions—Chapter 21) or membrane potential (Probes for Membrane Potential—Chapter 22) that can be followed with appropriately responsive fluorescent indicators. This chapter discusses Molecular Probes reagents and assays for detecting these diverse cell processes in live cells. Many of the assays in this chapter can be analyzed on a cell-by-cell basis and some are equally suitable for detection with a fluorescence microscope, flow cytometer or microplate reader. Almost all of the assays have the capacity for high-throughput analysis.
Our viability and cytotoxicity assay reagents (Viability and Cytotoxicity Assay Reagents—Section 15.2) and kits (Viability and Cytotoxicity Assay Kits for Diverse Cell Types—Section 15.3) are principally used to enumerate the proportion of live and dead cells in a population. In contrast, proliferation assays such as the Click-iT EdU cell proliferation assay (Assays for Cell Enumeration, Cell Proliferation and Cell Cycle—Section 15.4) are primarily designed to monitor the growth rate of a cell population or to detect daughter cells in a growing population. Fluorescence-based cell viability and proliferation assays are generally less hazardous and less expensive than radioisotopic techniques, more sensitive than colorimetric methods and more convenient than animal testing methods. Unlike 51Cr-release assays, fluorescence-based assays of cell-mediated cytotoxicity do not require large samples, which can be difficult to obtain. Furthermore, fluorescence-based protocols are more convenient than the trypan blue–based exclusion assay. This common colorimetric method for determining cell viability must be completed within 3–5 minutes because the number of blue-staining cells increases with time after addition of the dye.
Fluorescent dye–based assays for cell viability and cytotoxicity are reliable and easy to perform. Our stand-alone reagents for these assays are described in Viability and Cytotoxicity Assay Reagents—Section 15.2, whereas our kits for viability and cytotoxicity analysis are discussed in Viability and Cytotoxicity Assay Kits for Diverse Cell Types—Section 15.3. Molecular Probes LIVE/DEAD Viability Assay Kits (Viability and Cytotoxicity Assay Kits for Diverse Cell Types—Section 15.3) give researchers a choice of viability/cytotoxicity assays suitable for bacteria, fungi or higher eukaryotic cells. Our LIVE/DEAD Reduced Biohazard Cell Viability Kit and LIVE/DEAD Fixable Dead Cell Stain Kits (Viability and Cytotoxicity Assay Kits for Diverse Cell Types—Section 15.3) permit the original viability status of a mixed-cell population to be determined following aldehyde fixation of the sample in order to kill pathogens or in preparation for antibody staining. In each case, these viability assay kits provide the reagents and a simple protocol for simultaneous quantitation of live and dead cells. We also offer several proliferation assay kits that enable researchers to rapidly monitor numbers of adherent or nonadherent cells based on the presence of newly replicated DNA, total nucleic acid content or total protein content (Assays for Cell Enumeration, Cell Proliferation and Cell Cycle—Section 15.4). Assays for Apoptosis and Autophagy—Section 15.5 focuses on our probes for monitoring apoptosis, including reagents for selectively detecting apoptotic cells based on their cell-permeability properties, as well as conjugates of annexin V phosphatidylserine-binding protein for detecting phosphatidylserine externalization. Additionally, our Premo Autophagy Sensors and other fluorescent probes are useful for examining the role of autophagy in normal and diseased cells.
In addition to our probes for cell viability, cell proliferation, apoptosis and autophagy, several of the reagents for live-cell function described in Probes for Cell Adhesion, Chemotaxis, Multidrug Resistance and Glutathione—Section 15.6 can be used to develop assays that measure a particular biochemical parameter of interest. There is a significant overlap between probes for cell viability and probes for live-cell functions. For example, fluorogenic esterase substrates are commonly used to detect viability and proliferation, as well as to monitor cell adhesion, apoptosis and multidrug resistance. Likewise, cell-permeant and cell-impermeant nucleic acid stains are widely applicable to many live-cell function assays. We have organized discussions in this chapter according to several commonly studied cell processes in order to highlight the many published applications for these probes and foster the development of new applications.
The diversity of live cells and their environments () makes it impossible to devise a single viability or enumeration assay applicable to all cell types. Because viability is not easily defined in terms of a single physiological or morphological parameter, it is often desirable to combine several different measures, such as enzymatic activity, membrane permeability and oxidation–reduction (redox) potential. Each assay method has inherent advantages and limitations and may introduce specific biases into the experiment; thus, different applications often call for different approaches.