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:
- Cells growing in culture
- CyQUANT XTT Cell Viability Assay (Cat. No. X12223)
- Cell culture medium
- 96- or 364-well plate
- Microplate reader
- CO2 incubator
Protocol
 1. 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 |
 2. 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 |
 3. Add 70 µL of the working solution to each well |
 4. Incubate at 37°C for 4 hours in a CO2 incubator |
 5. 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
Click image to enlarge
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.