Microplate assays provide information on whole cell populations rather than tracking the behavior of individual cells. Life Technologies offers microplate assays for whole cells and assays performed on disrupted cells or cell lysates. Metabolic activity is commonly used as a viability indicator, but for some applications it can be important to assess viability independent of metabolic state and appropriate assays are listed below. In some cases, cells are required for additional downstream functional analysis and alamarBlue® is an excellent non-toxic indicator of viability.
Measuring Cellular Reduction Potential
The inherent reducing power of live cells serves as an indicator of metabolic activity and thus cell viability; when cellular reduction potential is measured in a population, the signal is proportional to the number of live cells. Some established assays such as MTT use that reducing power to generate a strongly colored reporter, formazan. Similarly, the redox indicator resazurin is converted to the fluorescent and colorimetric reporter molecule resorufin in metabolically active cells and it serves as a very reliable and sensitive viability/cytotoxicity indicator.
Life Technologies makes the assays simple and convenient by providing the reagents in a proprietary stabilizing formulation, with a mix, incubate, and read protocol that is scalable from single wells to high-throughput screening (HTS).
Monitoring the efficacy of a growth inhibitor with alamarBlue® indicator.
Measuring Membrane Permeability
The loss of plasma membrane integrity is a key marker of cell death that is regularly exploited by tools that measure viability and cytotoxicity. Cells with compromised plasma membranes allow the influx of cell-impermeant DNA-binding dyes that fluoresce only when bound to DNA. Thus, DNA staining can be used as a cytotoxicity indicator that is independent of metabolic state. Cytotoxic compounds can damage cells through a range of mechanisms. Cell viability, membrane integrity, and DNA content are among the most specific and sensitive parameters for measuring cytotoxicity and provide excellent sensitivity and accuracy.
A richer result is generated when DNA binding is combined with measurements of other parameters. Probes for esterase activity or cellular reduction potential stain live cells and provide measures of viability. A cell-permeant DNA stain will also stain cells with intact plasma membranes and serve as a marker for viable cells.
Dose response for valinomycin in HeLa cells using the HCS LIVE/DEAD® Green Kit.
Cell viability can be determined using microplate assays to measure indicators metabolic activity.
|Target||Measures cellular reduction potential where signal is proportional to the number of live cells|
|Readout||Fluorescence or absorbance readout||Colorimetric readout in cell lysates|
|Fluorescent label||Resazurin reduced to resorufin||XTT/Formazan||MTT/Formazan|
|Format||25 mL||100 mL||100 mg||1 kit/10 plates|
Cell viability can be determined using microplate assays to measure indicators metabolic activity or esterase activity. Cell membranes increase their permeability in damaged and dying cells allowing the influx of dyes which can both be measured using fluorescence changes. Combination assays measure multiple parameters to provide more context for determining the status of both live and dead cells.
|Target||Detects membrane permeability using fluorescent DNA binding dye(s)|
|Single-color assay||Two-color assay||Also measures metabolic activity||Also measures esterase activity|
|Readout||Dead cells fluoresce green||Intact cells emit mostly green, and dead cells emit mostly red||Dead cells emit mostly green, and active cells emit mostly red||Active cells emit mostly green, and dead cells emit mostly red|
|Fluorescent label||Image-iT® DEAD Green™||Image-iT® DEAD Green™||C12-resazurin||Calcein-AM|
|NuclearMask™ Deep Red or Hoechst 33342||SYTOX® Green||Ethidium homodimer-1|
|638/686 or 350/461||504/523||528/617|
|Format||1 vial/25 plates||1 kit/2 plates||1 kit/10 plates||1 kit/10 plates|
|Learn more from the Molecular Probes® Handbook