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XTT Cell Viability Assay Protocol

Specific for: XTT Cell Viability Kit #9095.

A. Reagent Preparation

  1. Thaw reagents just before experiment.

NOTE: Precipitation may occur when reagents are stored at −20°C. Make sure reagents are clear prior to use. If necessary, warm reagents to 37°C to reconstitute.

  1. Add electron coupling solution to XTT Reagent (1:50 volume ratio) to make XTT detection solution. For example, each 96-well plate needs 5 ml XTT solution and 0.1 ml electron coupling solution.

B. XTT Assay

  1. Add 50 µl XTT detection solution to each well of 96-well plate (which contains 100–200 µl/well culture medium) and return plate to incubator.
  2. Read absorbance at 450 nm.

NOTE: The optimal incubation time for this assay depends on experimental setup, such as: cell type, cell number, and treatment. Optimization of incubation time can be determined by reading one plate at various time points after addition of XTT detection solution (featured below).

Figure 1. C2C12 cells were seeded at varying density in a 96-well plate and incubated overnight.

posted October 2012