Microplate Assays for Viability Confirmation

What to do when ATP assays leave you guessing

Luciferase-based viability reporters, which measure ATP levels in the cell, will generate false viability readouts when a compound interferes directly with the Fluc enzyme. Several publications report how differences in a compound’s mechanism of action and cell line–specific responses can yield significantly misleading results when using ATP reporters [1–5].

Orthogonal (multiplexed) assay approaches using reporters with different modes of action (i.e., membrane permeability and cellular reduction potential) are generally used to provide a more stringent viability determination than ATP concentration.

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Multiplexed assay for reliable confirmation

The multiplexed PrestoBlue® and CyQUANT® Direct workflow is ideal for automation. Because the workflow measures unrelated orthogonal parameters, the data are more robust than a single assay. The combined assay can be performed at lower cost than a typical luminescent ATP assay, and can be readily combined with other fluorescent probes to provide further multiplexing and more data content.

By combining the PrestoBlue® and CyQUANT® Direct Assays into a single workflow, you can have reliable, orthogonal assay results in less than 90 minutes. The multiplex assay is easily automated, and dual reporters provide a richer understanding of your compound’s activity

  • HTS-compatible, with two additions and two reads (see protocol)
  • No cell lysis, wash steps, or media removal required (see protocol)
  • As fast as 70 minutes of total assay time
  • Robust signal—stable for up to 7 hours
  • More stringent than any individual viability reporter
  • Validated against multiple cell types (Figure 1)
  • Validated against multiple compound types (Figure 2)

Figure 1. PrestoBlue®/CyQUANT® Direct multiplex CellTiter-Glo® single readout comparison.

Figure 2. PrestoBlue®/CyQUANT® Direct multiple readouts.

PrestoBlue® Assay

The PrestoBlue® Assay is supplied ready to use and reports on cell viability based on the metabolic activity of the cell. 

  • HTS-compatible, addition-only, single-step assay protocol (see protocol)
  • No cell lysis, wash steps, or media removal required (see protocol)
  • Stable signal after 10 minutes of incubation time (Figure 3)
  • Linear dynamic range of 3 orders (Figure 3)
  • Robust assay readout stable for 8 hr (Figure 4)
  • Validated against multiple cell types (Figure 5)
  • Validated against multiple compound types (Figure 6)

Figure 3.
PrestoBlue® exhibits linear response over 3 orders of magnitude in culture media with one-step addition and 10 minute incubation time.

Figure 4.
Signal can be read at any time over a 7 hour period providing for a robust and flexible workflow in automated assays.

Figure 5. PrestoBlue® assays are tested of over a wide range of drug types and compound activities with similar results to CellTiter®-Glo.

Figure 6. PrestoBlue® and CellTiter®-Glo single readout comparison.

CyQUANT® Direct Assay

The CyQUANT® Direct Assay measures the number of viable cells with intact plasma membranes.

  • HTS-compatible, addition-only, single-step assay protocol (see protocol)
  • No cell lysis, wash steps, or media removal required (see protocol)
  • Stable signal after 60 minutes of incubation time (Figure 7)
  • Linear dynamic range of 3 orders (Figure 8)
  • Robust assay readout stable for 7 hr (Figure 9)
  • Validated against multiple cell types (Figure 10)
  • Validated against multiple compound types (Figure 11)

Figure 7. Linear readout over 30 minute to 90 minute incubation time.

Figure 8. Cell number sensitivity measured by cell type.

Figure 9. Stable readout over 60 minute to 7 hour incubation period.

Figure 10. Validated against multiple cell types.

Figure 11. Validated against multiple compound types.


  1. Firefly luciferase in chemical biology: a compendium of inhibitors, mechanistic evaluation of chemotypes, and suggested use as a reporter. Thorne N, Shen M, Lea WA, Simeonov A, Lovell S, Auld DS, Inglese J, Chem Biol. 2012; 19(8):1060–72
  2. A simple high-content cell cycle assay reveals frequent discrepancies between cell number and ATP and MTS proliferation assays. Chan GKY, Kleinheinz TL, Peterson D, Moffat JG, PLoS ONE 2013; 8(5):e63583
  3. Firefly luciferase inhibition. Leitao JM, Esteves da Silva JC, J Photochem Photobiol. 2010; 101(1):1–8
  4. Apparent activity in high-throughput screening: origins of compound-dependent assay interference. Thorne N, Auld D, Inglese J, Curr Opin Chem Biol. 2010; 14(3):315–324
  5. Characterization of chemical libraries for luciferase inhibitory activity. Auld DS, Southall NT, Jadhav A, Johnson RL, Diller DJ, Simeonov A, Austin CP, Inglese J, J Med Chem. 2008; 51(8):2372–86
Thermo Scientific plate readers

Expertly detect fluorescence with Thermo Scientific plate readers

High-sensitivity fluorescence detection for 96-1,536 samples can be quickly performed on Fluoroskan or Fluoroskan FL Microplate Fluorometer or Varioskan LUX Multimode Microplate Reader using Invitrogen reagents for optimal detection. Take advantage of automatic dynamic range selection to get optimal gain settings for each individual well and automation capabilities for even higher throughput.

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