PrestoBlue and CyQUANT Direct Confirmation Assay for Cell Viability Protocol

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:

Treated test cells growing in appropriate microplates
CyQUANT Direct Cell Proliferation Assay (Cat. No. C35011)
PrestoBlue Cell Viability Reagent (25 mL (Cat. No. A13261) or 100 mL (Cat. No. A13262))
Phosphate-buffered saline (PBS) (Cat. No. 14190-144)
Fluorescence microplate reader with bottom-read mode

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	560/590 nm	485/538 nm
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

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

Differences in the EC₅₀/IC₅₀determinations 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.