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.