image of CyQuant® Direct Assay Kit and PrestoBlue® reagent bottle

Two-parameter cell viability assay

The PrestoBlue® and CyQUANT® Direct confirmation assay uses orthogonal assay mechanisms to provide a more stringent measure of viability than any single assay parameter. PrestoBlue® reagent responds to the reducing environment of viable cells and the CyQUANT® Direct assay reports on the number of live cells with intact cell membranes.

The combined assay workflow is optimized for automation in 96-well or 384-well microplates using a bottom-read fluorescence plate reader.

This protocol can be used for:

  • Quantifying live cells using a fluorescence microplate reader

You will need the following for this protocol:

Assay protocol

1. Pre-incubate or dispense cells in appropriate microplate wells
2. Add PrestoBlue® 10X reagent to microplate wells (see recommended volumes)
3. Incubate at 37°C for 10 minutes
4. Read fluorescence at 590 nm
5. Add CyQUANT® Direct 2X detection reagent to microplate wells (see instructions for making 2X detection reagent and recommended well volumes)
6. Incubate at 37°C for 60 minutes
7. Read fluorescence at 538 nm
7. Plot a curve of relative fluorescence units vs. drug concentration to generate quantitative results for both assays
Spectral information and storage
  PrestoBlue® 10X Cell Viability Reagent CyQUANT® Direct 2X detection reagent
Excitation/Emission (in nm) 560/590 485/538
Storage conditions 4°C 4°C


Protocol tips

  • The assay can tolerate a variety of cell culture media components including phenol red and up to 10% serum
  • When prepared aseptically, the CyQUANT® Direct 2X detection reagent is generally stable for up to 24 hours at room temperature

Table 1. PrestoBlue® reagent recommended volumes.

PrestoBlue® Cell Viability Reagent is supplied as a 10X solution. Bring PrestoBlue® Reagent to room temperature and add directly to cells in culture medium. See table below for example volumes. PrestoBlue® signal is stable for 7 hours.

Format Volume of cells + medium Volume of PrestoBlue® reagent
96-well plate 90 μL 10 μL
384-well plate 36 μL 4 μL
1,536-well plate 5 μL 3 μL

Making 2X CyQUANT® Direct Detection Reagent solution

1. Combine the following in a 15 mL tube:

  • Hank’s buffered saline solution, PBS, or cell culture medium—11.7 mL
  • CyQUANT® Direct nucleic acid stain—48 µL
  • CyQUANT® Direct background suppressor I—240 µL

2. Mix well and let stand at room temperature until ready for use. See below for example volumes. CyQUANT® Direct signal is stable for 7 hours

Table 2. CyQUANT® Direct reagent recommended volumes.

Format Volume of cells + medium/well Volume of 2X detection reagent/well
96-well plate 100 μL 100 μL
384-well plate 40 μL 40 μL
1,536-well plate 5 μL 2 μL

Notes on data analysis for orthogonal assays

  1. Subtract background reading from all sample well results
  2. Use a nonlinear regression analysis of log(inhibitor) vs. response to determine EC50/IC50 value
  3. If raw response values are used, plot on dual y-axes
  4. If normalized response values are used, plot on the same axis

Differences in the EC50/IC50 determinations between PrestoBlue® and CyQUANT® Direct reagents reflect differences in the assay mechanism. Such orthogonal results may provide insight into the mode of action of a compound.