image of CyQUANT® Direct Microplate Reagent Kit

Cell membrane integrity—based viability assay

The CyQUANT Direct assays are based on a cell-permeant DNA-binding dye in combination with a background suppression reagent. The DNA-binding dyes stains all cells, but in cells with compromised cell membranes the fluorescence is masked by the background suppressors. Thus, the fluorescent signal in the CyQUANT Direct assays are proportional to the number of live cells. Live cells can also be counted using an imaging system. The assays are available in either green or red emission versions.

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:

  • Treated test cells growing in appropriate microplates
  • CyQUANT Direct Cell Proliferation Assay (Cat. No. C35011 & C35012)
  • CyQUANT Direct Red Cell Proliferation Assay (Cat. No. C35013 & C35014)
  • Phosphate-buffered saline (Cat. No. 14190-144)
  • Fluorescence microplate reader with bottom-read mode

Assay protocol

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1. Pre-incubate test cells in appropriate microplate wells

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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)

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3. Incubate for 60 minutes at 37°C

4. Read fluorescence using excitation/emission
  • CyQUANT Direct—508/527 nm
    CyQUANT Direct Red—622/645 nm

    Note: Signal is stable for 7 hours.
Plot

5. Plot a standard curve of fluorescence vs. cell number to generate quantitative results

 

 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.