image of CyQUANT® Direct Microplate Reagent Kit

Cell membrane integrity—based viability assay

The CyQUANT® Direct assay is based on a cell-permeant DNA-binding dye in combination with a background suppression reagent. The DNA-binding dye stains all cells, but in cells with compromised cell membranes the fluorescence is masked by the background suppressor. Thus, the fluorescent signal in the CyQUANT® Direct assay is proportional to the number of live cells. Live cells can also be counted using an imaging system.

This protocol can be used for:

  • Quantifying live cells using a microplate reader

This protocol should not be used for:

  • Flow cytometry

You will need the following for this protocol:

Assay protocol

1. Pre-incubate test cells in appropriate microplate wells
2. Add an equal volume of CyQUANT® Direct 2X detection reagent to microplate wells (see instructions for making 2X detection reagent and recommended well volumes)
3. Incubate for 60 minutes at 37°C
4. Read fluorescence at 538 nm (signal is stable for 7 hours)
5. Plot a standard curve of fluorescence vs. cell number to generate quantitative results
Spectral information and storage

CyQUANT® Direct Cell Proliferation Assay

Excitation/Emission (in nm) 508/527
Storage conditions Frozen or ambient temperature for up to 6 months


Protocol tips

  • The assay can tolerate a variety of cell culture media components including phenol red and up to 10% serum
  • Plot a standard curve of fluorescence vs. cell number to generate quantitative results
  • When prepared aseptically, the 2X detection reagent is generally stable for up to 24 hours at room temperature
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— 240 µL

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

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
For Research Use Only. Not for use in diagnostic procedures.