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.

Explore our portfolio of easy-to-learn, easy-to-use plate readers


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)

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)

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)
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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