formazan

Formazan is the general name for colored dyes produced when certain tetrazolium salts are reduced. In laboratory assays, formazan dyes serve as indicators of cellular metabolic activity and are widely used to assess cell viability, proliferation, and cytotoxicity. The color and solubility of the formazan depend on the substrate employed.

The most common substrates are MTT, XTT, WST-1, WST-8, and MTS. In viable cells, mitochondrial and other dehydrogenases transfer electrons from donors such as NADH or NADPH to the tetrazolium salt, reducing it to a formazan product. With MTT, the formazan is insoluble and forms purple crystals that accumulate in the cells and require a solubilization step (often with DMSO or isopropanol) before measuring absorbance. XTT, WST-1, and WST-8 typically yield water-soluble formazans, enabling direct spectrophotometric readout. MTS also produces a soluble formazan under standard conditions. The color generally ranges from purple (MTT) to orange (XTT/WST). Wavelengths for readout vary by reagent, commonly around 570 nm for MTT and about 450–490 nm for soluble-formazan assays.

Applications and interpretation: These assays provide a relative measure of viable cell number or metabolic activity, with absorbance proportional to the amount of formazan formed under controlled conditions. They are popular due to simplicity, speed, and scalability for high-throughput screening.

Limitations: results can be affected by non-specific reducing agents, assay timing, solvent effects (for insoluble forms), and variations in cellular metabolism. Consequently, formazan-based assays are most reliable when carefully validated for the specific cell type and experimental conditions.