Protocols and Applications Guide (US Letter Size) - Promega

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|||| 4Cell Viability I. Introduction Choosing a cell viability or cytotoxicity assay from among the many different options available can be a challenging task. Picking the best assay format to suit particular needs requires an understanding of what each assay is measuring as an endpoint, of how the measurement correlates with cell viability, and of what the limitations of the assay chemistries are. Here we provide recommendations for characterizing a model assay system and some of the factors to consider when choosing cell-based assays for manual or automated systems. A. Establishing an In Vitro Model System The species of origin and cell types used in cytotoxicity studies are often dictated by specific project goals or the drug target that is being investigated. Regardless of the model system chosen, establishing a consistent and reproducible procedure for setting up assay plates is important. The number of cells per well and the equilibration period prior to the assay may affect cellular physiology. Maintenance and handling of stock cultures at each step of the manufacturing process should be standardized and validated for consistency. Assay responsiveness to test compounds can be influenced by many subtle factors including culture medium surface-to-volume ratio, gas exchange, evaporation of liquids and edge effects. These factors are especially important considerations when attempting to scaleup assay throughput. B. Choosing an Endpoint to Measure One of the first things to decide before choosing an assay is exactly what information you want to measure at the end of a treatment period. Assays are available to measure a variety of different markers that indicate the number of dead cells (cytotoxicity assay), the number of live cells (viability assay), the total number of cells or the mechanism of cell death (e.g., apoptosis). Table 4.1 compares Promega homogeneous cell-based assays and lists the measured parameters, sensitivity of detection, incubation time and detection method for each assay. A basic understanding of the changes that occur during different mechanisms of cell death will help you decide which endpoint to choose for a cytotoxicity assay (Riss and Moravec, 2004). Figure 4.1 shows a simplified example illustrating chronological changes occurring during apoptosis and necrosis and the results that would be expected from using the assays listed in Table 4.1 to measure different markers. Protocols & Applications Guide www.promega.com rev. 8/06 Figure 4.1. Mechanisms of cell death can be determined by measuring different markers of cell viability and apoptosis in vitro. Cultured cells that are undergoing apoptosis in vitro eventually undergo secondary necrosis. After extended incubation, apoptotic cells ultimately shut down metabolism, lose membrane integrity and release their cytoplasmic contents into the culture medium. Markers of apoptosis such as caspase activity may be present only transiently. Therefore, to determine if apoptosis is the primary mechanism of cell death, understanding the kinetics of the cell death process in your model system is critical. Cells undergoing necrosis typically undergo rapid swelling, lose membrane integrity, shut down metabolism and release their cytoplasmic contents into the surrounding culture medium. Cells undergoing rapid necrosis in vitro do not have sufficient time or energy to activate apoptotic machinery and will not express apoptotic markers. [For additional information about mechanisms of cell death, please visit the Apoptosis Chapter (www.promega.com /paguide/chap3.htm) of this Protocols and Applications Guide.] If the information sought is simply whether there is a difference between “no treatment” negative controls and “toxin treatment” of experimental wells, the choice between measuring the number of viable cells or the number of dead cells may be irrelevant. However, if more detailed information on the mechanism of cell death is being sought, the duration of exposure to toxin, the concentration of the test compound, and the choice of the assay endpoint become critical (Riss and Moravec, 2004). C. Characterizing Assay Responsiveness Protocols used to measure cytotoxicity in vitro differ widely. Often assay plates are set up containing cells and allowed to equilibrate for a predetermined period before adding test compounds. Alternatively, cells may be added PROTOCOLS & APPLICATIONS GUIDE 4-1
|||| 4Cell Viability Table 4.1. Comparison of Promega Cell Viability, Cytotoxicity and Apoptosis Assays CellTiter 96® AQueous One Solution Cell Proliferation Assay 1–4 hours MTS reduction 800 cells/200 cells CellTiter-Blue® Cell Viability Assay 1–4 hours resazurin reduction 390 cells/50 cells BacTiter-Glo® Microbial Cell Viability Assay 5 minutes ATP ~40 cells/N.D. suspension or adherent cells colorimetric suspension or adherent cells fluorometric or colorimetric bacteria, yeast luminescent CellTiter-Glo® Luminescent Cell Viability Assay 10 minutes ATP 50 cells/15 cells (also 1536-well format) suspension or adherent cells luminescent Characteristic Incubation Parameter measured Sensitivity: 96-well/384-well Sample Type Detection Protocols & Applications Guide www.promega.com rev. 8/06 Caspase-Glo® 8 or 9 Assays 30 minutes–2 hours initiator caspase activity 20 cells/20 cells culture cells or purified enzyme luminescent Caspase-Glo® 3/7 Assay 30 minutes–2 hours effector caspase activity 20 cells/20 cells culture cells or purified enzyme luminescent Apo-ONE® Homogeneous Caspase-3/7 Assay 1–18 hours effector caspase activity Several hundred cells in a population culture cells or purified enzyme fluorometric Characteristic Incubation Parameter measured Sensitivity: 96-well/384-well Sample Type Detection CytoTox 96® Non-Radioactive Cytotoxicity Assay 30 minutes LDH Release CytoTox-Fluor Cytotoxicity Assay 30 minutes dead-cell protease activity MultiTox-Fluor Multiplex Cytotoxicity Assay 30 minutes live- and dead-cell protease activity several hundred cells or cell equivalents (also in 1536-well format) suspension or adherent cells fluorometric CytoTox-ONE Membrane Integrity Assay 10 minutes LDH release Characteristic Incubation Parameter measured several hundred cells or cell equivalents several hundred cell equivalents 800 cells/200 cells Sensitivity: 96-well/384-well suspension or adherent cells colorimetric suspension or adherent cells fluorometric suspension or adherent cells fluorometric Sample Type Detection PROTOCOLS & APPLICATIONS GUIDE 4-2
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CONTENTS I. Nucleic Acid Amplificat
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Table 8.1. pGL4 Luciferase Reporter
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Protocols and Applications Guide (US Letter Size) - Promega

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