Isolation and High Throughput Flow Cytometric Apoptosis Assay of Human Neutrophils to Enable Compound Library...

Area of Science:

• Immunology
• Cell Biology
• Pharmacology

Background:

• Studying human neutrophils in vitro is difficult due to their short lifespan and tendency to activate.
• Neutrophils play a key role in immune responses, making their study crucial for understanding health and disease.

Purpose of the Study:

• To describe a reproducible method for isolating human neutrophils from peripheral blood.
• To establish a high-throughput screening protocol to identify compounds that induce neutrophil apoptosis.
• To explore therapeutic potential for neutrophil-driven diseases.

Main Methods:

• Neutrophil isolation using Dextran sedimentation and Percoll gradient centrifugation.
• Treatment of isolated neutrophils with a library of kinase inhibitors.
• Assessment of neutrophil apoptosis rates via flow cytometry.

Main Results:

• Achieved typical yields of ~1 x 10⁶ neutrophils/mL with >95% purity.
• Successfully established a protocol for high-throughput screening of primary human immune cells.
• Identified compounds capable of modulating neutrophil apoptosis.

Conclusions:

• The described protocol provides a reliable method for human neutrophil isolation and functional screening.
• This approach facilitates the discovery of potential therapeutic agents targeting neutrophil-mediated inflammatory conditions.
• The protocol is adaptable for assessing other neutrophil functions.