96-well plate assay for sublethal metabolic activity

Area of Science:

• Biochemistry
• Cell Biology
• Toxicology

Background:

• Assessing compound effects on cellular metabolism and viability is crucial for drug discovery.
• Existing methods can be time-consuming and expensive for large-scale screening.

Purpose of the Study:

• To develop and validate a 96-well plate assay for estimating sublethal metabolic activity and cytolethality of compounds in vitro.
• To screen five glycolysis inhibitors to demonstrate the assay's utility in assessing metabolic inhibition without causing cell death.

Main Methods:

• Combined resazurin reduction, lactate production, and glucose consumption assays.
• Exposed in vitro fibroblast cell cultures to five glycolysis inhibitors (phloretin, 2-deoxyglucose, iodoacetate, fluoride, oxamate) for 6 and 24 hours.
• Determined IC(50) values for metabolic inhibition and cytolethality.

Main Results:

• Iodoacetate was the most potent glycolysis inhibitor and cytolethal agent within 6-hour exposures.
• 2-Deoxyglucose exhibited the broadest sublethal metabolic range.
• The assay successfully differentiated between metabolic inhibition and cell death for tested compounds.

Conclusions:

• The developed assay provides a simple, inexpensive, and efficient method for evaluating global metabolic and cytolethal effects of compounds in vitro.
• This assay is valuable for large-scale screening and ranking of compounds in early drug discovery and development.
• It serves as a cost-effective initial step before employing more complex in vitro methods.