Antibody-dependent cell-mediated cytotoxicity: a flow cytometry-based assay using fluorophores

Area of Science:

• Immunology
• Cell Biology
• Biotechnology

Background:

• Antibody-dependent cell-mediated cytotoxicity (ADCC) is a critical immune mechanism.
• Assessing ADCC traditionally involves radiochemicals, posing safety and handling challenges.
• A need exists for a safer, more precise method to quantify ADCC.

Purpose of the Study:

• To develop and validate a novel flow cytometry-based assay for determining ADCC.
• To offer a non-radioactive alternative for ADCC measurement.
• To establish a reliable method for evaluating antibody therapeutics.

Main Methods:

• Target cells were labeled with the membrane dye PKH-26.
• Incubation of labeled target cells with effector cells and antibody.
• Assessment of target cell death using the viability probe TO-PRO-3 iodide (TP3) via flow cytometry.
• Analysis of cell death on a single-cell basis within the PKH-26+ population.

Main Results:

• The developed assay allows for precise discrimination of target cells.
• Cell death within the target cell population was accurately quantified.
• The assay demonstrated accuracy and reproducibility in ADCC determination.
• The method successfully avoids the use of hazardous radiochemicals.

Conclusions:

• A novel, non-radioactive flow cytometry assay for ADCC determination has been successfully developed.
• This assay provides accurate, reproducible, single-cell level analysis of ADCC.
• The method holds significant potential as a valuable tool for assessing antibody-based therapeutics.