Resazurin Assay Protocol

Resazurin is a cell-permeable redox indicator that can be used to monitor viable cell numbers with protocols similar to those utilizing the tetrazolium compounds. Resazurin can be dissolved in physiological buffers (resulting in a deep blue-colored solution) and added directly to cells in culture in a homogeneous format. Viable cells with active metabolism can reduce resazurin into the resorufin product which is pink and fluorescent.

The addition of an intermediate electron acceptor is not required for cellular resazurin reduction to occur, but it may accelerate signal generation. The quantity of resorufin produced is proportional to the number of viable cells which can be quantified using a microplate fluorometer equipped with a 560 nm excitation / 590 nm emission filter set. Resorufin also can be quantified by measuring a change in absorbance; however, absorbance detection is not often used because it is far less sensitive than measuring fluorescence. The resazurin reduction assay is slightly more sensitive than tetrazolium reduction assays and there are numerous reports using the resazurin reduction assay in a miniaturized format for HTS applications.

Reagent preparation

• Dissolve high-purity resazurin in DPBS (pH 7.4) to 0.15 mg/ml.
• Filter-sterilize the resazurin solution through a 0.2 μm filter into a sterile, light-protected container.
• Store the resazurin solution protected from light at 4°C for frequent use or at -20°C for long-term storage.

Assay protocols

• Prepare cells and test compounds in opaque-walled 96-well plates containing a final volume of 100 µl/well. An optional set of wells can be prepared with medium only for background subtraction and instrument gain adjustment.
• Incubate for a desired period of exposure.
• Add 20 µl resazurin solution to each well.
• Incubate 1 to 4 hours at 37°C.
• Record fluorescence using a 560 nm excitation / 590 nm emission filter set.

Creative Bioarray Relevant Recommendations

• Creative Bioarray offers a wide selection of cell viability assays for drug screening and cytotoxicity tests of chemicals. We offer a variety of reagents for studying cell viability including fluorescent and non-fluorescent reporter dyes.

• Determine the optimal concentration of Resazurin and the appropriate incubation time through preliminary optimization experiments to ensure accurate and reproducible results.
• Follow the manufacturer's guidelines for storing and handling Resazurin reagents to prevent degradation and ensure their effectiveness.
• Validate the Resazurin assay with alternative assays, such as trypan blue exclusion or live/dead staining, to confirm the reliability of the viability measurements.

Cell Viability, Proliferation, and Cytotoxicity

Cell Viability Assays

Cell Line Testing and Assays