CyQuant XTT Product packaging

Metabolic activity-based cell viability assay

XTT is a two-reagent kit that consists of the XTT reagent and the Electron Coupling Reagent. This is a colorimetric assay used to assess cell viability as a function of cell number based on metabolic activity. The water-soluble XTT is converted to an orange-colored formazan product with an absorbance read at 450 nm and 660 nm.

This protocol can be used for:

  • Quantification of cell number based on metabolic activity in microplate readers capable of absorbance measurements

This protocol should not be used for:

  • Fluorescence microscopy

You will need the following for this protocol:

Protocol

cell culture plate1. Seed cells in a 96-well plate containing 100 μL/well of culture medium with compounds to be tested. Culture in a CO2 incubator for 24–48 hours
tube with liquid2. Add 1 mL of Electron Coupling Reagent to 6 mL of XTT Reagent; vortex to mix. Once prepared, this working solution should be used promptly
liquid being added to cell culture plate3. Add 70 µL of the working solution to each well
timer4. Incubate at 37°C for 4 hours in a CO2 incubator
cell culture plate getting analyzed5. Read absorbance at 450 nm and 660 nm

Protocol tips

  • Immediately use the XTT/Electron Coupling Reagent after preparation
  • Freeze/thaw cycles will affect assay performance, as a result single-use vials are provided to ensure stability and should be kept at –20ºC until use
  • Washing is not required after staining
  • To correct for background absorbance, include at least three wells with only complete culture medium without cells
  • A cell titration experiment using a range of cells from 103–105 cells/well can be used to generate a standard curve

Figure 1. A549 cells were assayed using either the CyQUANT XTT Cell Viability Assay or a commercially available MTT according to their respective manufacturers’ protocols. The signal-to-background ratio for the MTT assay product is calculated as the absorbance of the sample at 540 nm divided by the absorbance of the blank at 540 nm.

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