Quantifying Neutrophil Extracellular Traps in Real Time

Neutrophils are the first line of defense at the site of an infection, playing an essential role in the innate immune system, employing multiple strategies to degrade and kill microbes including the release of neutrophil extracellular traps (NETs). NETs are a web-like structure composed of antimicrobial proteins and DNA that are released during a distinct form of programmed neutrophil cell death known as NETosis. NETs are a host-defense strategy that results in the trapping and subsequent degradation of pathogens, but have been implicated in a variety of autoimmune diseases, including rheumatoid arthritis, as well as pathological conditions.

Our strategy

Using the Incucyte® Live-Cell Imaging System and the Incucyte® Cytotox Green Reagent allows automatic quantification of neutrophils undergoing NETosis in real time. Using Incucyte® live-cell reagents and analysis, the NETosis stages of swelling and decondensation of nuclei, followed by NET release, can be observed and measured.

NETosis Assay

Introducing Incucyte® NETosis Assay

An integrated solution to automatically visualize and quantify NETosis, in real time and in 96-well formats — inside your tissue culture incubator.

Measure NETosis using the IncuCyte® Live-Cell NETosis assay. Real-time detection of NETosis in human neutrophils treated with PMA (left). Neutrophils undergoing NETosis are labeled green using the mix-and-read IncuCyte Cytotox Green Reagent and quantified in real time using the IncuCyte Live-Cell Analysis System.

Key Advantages

Validate NETosis using time-lapse imaging

Incucyte® live-cell analysis allows for validation of the distinct morphological changes associated with NETosis - follow the loss of multilobulated nuclei, nuclear decondensation and membrane compromise in real time.

Validate NETosis using IncuCyte HD images.  IncuCyte images of  DMSO (1.25% v/v) and ATRA (0.1 µM) differentiated HL-60 cells stimulated using PMA in the presence of IncuCyte Cytotox Green reagent. Note the characteristic decondensation of the nuclei ~2 hours post stimulation. As cytoplasm mixes with karyoplasm, the nuclei are no longer visible in phase images. The nuclear contents are moved to the plasma membrane and released, with external DNA binding to the IncuCyte Cytotox Green Reagent, and fluorescent enhancement (green) is observed.

Automated analysis over the entire assay time course using Incucyte Cytotox Green

Determine how and when treatment effects occurred without removing cells from the stable environment of the incubator - ideal for sensitive cells.

Quantify treatment effects automatically and non-invasively. The IncuCyte NETosis assay allows every well of a 96-well plate to be imaged and analyzed (fluorescent images) automatically to provide a kinetic readout of neutrophil physiology over time (left graph). Using the integrated Incucyte® basic analyzer software with area filters (right graph, excluding objects < 300 µm2) gives the ability to discriminate between apoptotic (CMP treated, smaller labeled cells, only nucleus becomes fluorescent with loss of viability) and NETosis (PMA treated, nuclear contents are moved to the plasma membrane and released, with external DNA being labeled) inducing treatments.

Simple mix-and-read 96 well protocols suitable for pharmacological screening; no washing, no fixing, no lifting

Utilizing the mix-and-read Incucyte Cytotox Green reagent, rapidly visualize and quantify NETosis as extracellular DNA is released and undergoes fluorescence enhancement.   

Automated analysis over the entire assay time course using Incucyte® Cytotox Green

Reveal concentration-dependent responses and conduct pharmacological analyses. dHL-60 cells were treated with increasing concentrations of the NADPH oxidase inhibitor diphenylene iodonium (DPI).  Cells were then stimulated with PMA and labeled with Incucyte Cytotox green using a simple mix-and-read protocol. The time course graph displays an increase in total green object area over time with 100 nM PMA which is inhibited at increasing concentrations of DPI (A); Plate views taken from IncuCyte show clear positive and negative control responses (row H) with concentration dependent responses for each Inhibitor (n=3 per inhibitor, per concentration) (B); Area under the curve analysis can be performed to display a clear concentration dependent response (C).

Multiplexing with apoptotic readouts enables mechanistic insights

Combine the Incucyte® Annexin V Red Reagent with the Incucyte Cytotox Green Reagent for multiplexed measurements - readily discriminate between apoptosis and NETosis.

Multiplex NETosis measurements with PS externalization. Cells stimulated with PMA or ionomycin labeled with Incucyte Cytotox Green Reagent (NETosis – bright fluorescence, large object area) or Incucyte Annexin V Red (PS externalization– low fluorescence) to detect differential neutrophil cell death.

Ordering Information

The Incucyte Cytotox Green and Annexin V Red Reagents are fully validated for use with the Incucyte Live-Cell Analysis System, and can be combined for multiplexed measurements of NETosis and apoptosis within the same well.

Technical Resources

Literature and Documentation


Incucyte® Reagents, Consumables and Software

Download Now

Protocol — Incucyte® NETosis Assay

Download now

Brochure — Assays for Immunology Research

Download now

Poster — Real-time visualization and quantification of neutrophil extr...

Download now

Related Applications

A High Throughput Real-Time Imaging Technique to Quantify NETosis and Distinguish Mechanisms of Cell Death in Human Neutrophils. 

A High Throughput Real-Time Imaging Technique to Quantify NETosis and Distinguish Mechanisms of Cell Death in Human Neutrophils. Gupta S, Chan DW, Zaal KJ, Kaplan MJ. J Immunol, 200: 869-879, 2017

“The Incucyte platform is a novel real-time assay that quantifies NETosis in a rapid, automated, and reproducible way, significantly optimizing the study of neutrophils. This platform is a powerful tool to assess neutrophil physiology and NETosis, as well as to swiftly develop and test novel neutrophil targets.”

Request a Quote, Demo, or More Information

What other areas are you interested in? (select all that apply)

Request a Demo, Literature, or More Information