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.
|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
|PrestoBlue 10X cell viability reagent
|CyQUANT Direct 2X detection reagent
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.
|Volume of cells + medium
|Volume of PrestoBlue reagent
1. Combine the following in a 15 mL tube:
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.
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.