Live Imaging and Quantification of Neutrophil Extracellular Trap Formation

Area of Science:

• Immunology
• Cell Biology
• Microscopy

Background:

• Neutrophil Extracellular Trap (NET) formation, or NETosis, is a critical immune response to various stimuli.
• Investigating NETosis traditionally requires complex methodologies and specialized software.
• There is a need for accessible and efficient methods to study NETosis.

Purpose of the Study:

• To develop and describe a fully automated assay for quantifying NETosis.
• To provide a user-friendly protocol that does not require complex programming or commercial software.
• To enable reproducible and straightforward analysis of NETosis.

Main Methods:

• Utilized fluorescence live imaging on an automated widefield inverted microscope.
• Developed a protocol encompassing sample preparation, automated acquisition setup, and data analysis.
• Employed the readily available image analysis software Fiji (ImageJ2) for quantification.

Main Results:

• Successfully established a fully automated assay for NETosis quantification.
• The protocol details sample preparation, automated imaging acquisition, and data analysis steps.
• The method is adaptable for various stimuli and can be modified for high-throughput analysis.

Conclusions:

• The described automated assay offers an easy-to-use method for investigating NETosis.
• This protocol facilitates the study of NETosis without specialized technical expertise or expensive software.
• The adaptable nature of this assay supports diverse research applications, including high-throughput screening.