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How might environmental factors affect the cell-based MultiTox-Fluor, MultiTox-Glo, CytoTox-Fluor™ and CytoTox-Glo™ Assays?

Promega offers four assays with different substrates to assess cellular health. The CytoTox-Glo™ Assay (Cat.# G9290), a luminescent cytotoxicity assay, and CytoTox-Fluor™ Cytotoxicity Assay (Cat.# G9260), a single-reagent-addition, homogeneous, fluorescent assay, measure the relative number of dead cells in cell populations. Both assays measure the extracellular activity of a distinct intracellular protease activity (dead-cell protease) when the protease is released from membrane-compromised cells. The MultiTox-Fluor Multiplex Cytotoxicity Assay (Cat.# G9200) is a single-reagent-addition, homogeneous, fluorescent assay that measures the number of live and dead cells simultaneously in culture wells, while the MultiTox-Glo Multiplex Cytotoxicity Assay (Cat.# G9270) is a sequential-reagent-addition fluorescent and luminescent assay that measures the relative number of live and dead cells in cell populations. Both multiplex assays measure two protease activities; one is a marker of viability, and the other is a marker of cytotoxicity. With a luminescent substrate, fluorescent substrate or both, there are many environmental factors that can influence how the assays work.

• Temperature: The generation of fluorescent and luminogenic products is proportional to the protease activity associated with cell viability and cytotoxicity. The activity of these proteases and the luciferase detection chemistry is influenced by temperature. For best results, we recommend incubating the assays at a constant temperature to ensure uniformity across the plate. Assays are incubated at 37°C for fluorescence or room temperature for luminescence or fluorescence. Incubation at 37°C in a CO₂ culture cabinet may lead to edge-effects, the result of thermal gradients across the plate where the edges equilibrate more quickly to the ambient temperature than the center. Fluorescent assays performed at room temperature may require more than 30 minutes of incubation; however, do not incubate longer than three hours. Luminescent assays can only be incubated at room temperature.
• Filter Sets and Instrumentation:
  Fluorescence substrates: Fluorogenic dyes exhibit distinct absorption (excitation) and emission profiles when a light energy source is applied. In multiplexed formats, there is often a modest overlap in these profiles beyond their optimal peaks. Most fluorometers (or multimode instruments) contain optical band-pass filters, which restrict the wavelengths of light used to excite a fluorophore and the wavelengths passing through to the detector. Note that deviation from the optimal filter set recommendations may adversely affect assay sensitivity, signal separation and performance.
  Luminescence substrates: Luminescence chemistry does not require an external light source to generate photons. The detection instrument should be set to collect unrestricted/unfiltered light output.
• Cytotoxicity Marker Half-Life: The activity of the dead-cell protease marker has a half-life estimated to be greater than 10 hours. In situations where cytotoxicity occurs very rapidly (necrosis) and the incubation time is greater than 24 hours, the dead-cell protease activity and thus the degree of cytotoxicity, may be underestimated. When using extended incubation times, adding a lytic detergent such as digitonin may be useful to determine the total cytotoxicity marker activity (from remaining live cells).
• Luminescent Signal Half-Life: The luminescent dead-cell assays (CytoTox-Glo™ Cytotoxicity Assay and part of the MultiTox-Glo Multiplex Assay) use the AAF-Glo™ Substrate in conjunction with a thermostable, recombinant luciferase to generate a “glow-type” luminescent signal that is proportional to the number of dead cells in the sample. After a short incubation (0–15 minutes), the signal reaches a plateau and is relatively stable for a period of approximately 1 hour. Although the signal half-life is greater than 5 hours, we suggest measuring the luminescent signal at steady-state conditions (15 minutes to 1 hour).
• Light Sensitivity: The MultiTox-Glo, MultiTox-Fluor and CytoTox-Fluor™ Assays use a fluorogenic peptide substrate to measure the number of live cells. Although the substrate demonstrates good general photostability, the liberated fluor (AFC) or aminoluciferin can degrade with prolonged exposure to ambient light sources. We recommend shielding the plates from ambient light at all times.

For more frequently asked questions on these assays and others, visit our FAQ Database.

Technical Information

• CytoTox-Glo™ Cytotoxicity Assay Technical Bulletin #TB359
• CytoTox-Fluor™ Cytotoxicity Assay Technical Bulletin #TB350
• MultiTox-Fluor Multiplex Cytotoxicity Assay Technical Bulletin #TB348
• MultiTox-Glo Multiplex Cytotoxicity Assay Technical Bulletin #TB358