Dear Dr. César Germán Prucca

Alamar blue assay involves reduction of resazurin, a non-fluorescent compound, to resorufin, a fluorescent compound, by metabolically active cells. Please note that there are some compounds that can directly interact with resazurin, either reducing it to resorufin (like a reducing agent might) or oxidizing it (like an oxidizing agent might), thus influencing the assay's fluorescence signal.

If your chemical compound is a reducing agent, it might cause excessive reduction of resazurin, leading to an overestimation of cell viability because you'll see more resorufin than you should based on cell metabolism alone. Conversely, if your chemical compound is an oxidizing agent, it might prevent resazurin from being reduced, leading to an underestimation of cell viability.

Since the microscope confirmation shows cell death, you should not observe fluorescence because dead cells lack the necessary metabolic activity to reduce resazurin, so they will not produce the pink, fluorescent resorufin. So, in addition to the present control (culture media mixed with the alamar blue reagent), it's crucial to include a control where resazurin is incubated with the chemical compound (but without cells) in culture medium. This control will help determine if the chemical compound itself reduces resazurin, causing a false positive result. Without this control, it would be impossible to distinguish between resazurin reduction due to the chemical compound and that due to cellular metabolism.

In conclusion, if the chemical compound interferes with resazurin reduction in alamar blue assay, alternative assays like MTT, XTT, or ATP-based assays can be used to assess cell viability. These assays are less susceptible to the compound's interference because they rely on different mechanisms for detecting cell viability.

Regards,

Malcolm Nobre

Thank you, Malcom, for your response. We have been using this reactive for quite some time and have recently encountered these problems. I would perform the control only with the compounds. Thank you again for this recommendation.

Best,

César