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Automated Analysis of Neutrophil NETosis ActivityDownload
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February 06, 2019
Authors: Paul Held, BioTek Instruments, Inc., Winooski, VT, USA and Julie Rumble, Cayman Chemical, Ann Arbor, MI, USA
Neutrophils play a key role in the body’s defense against infections. As part of the innate immune system, they employ 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. In general, NETs allow neutrophils to kill extracellular pathogens while minimizing damage to the host cells, but can also result in an autoimmune response. This process in vivo is not well understood, and the development of in vitro models allows for further elucidation of the mechanisms and results of this phenomenon.
We have used differentiated HL-60 (dHL60) cells as an in vitro model for human neutrophils. dHL60 cells exhibit the same NETosis response as freshly isolated human cells and serve as a useful and easy to culture model to better understand this phenomenon. NETosis was examined using imaging after staining with a combination of nuclear dyes. While both dyes bind nucleic acids and become fluorescent, one is cell membrane permeant that can stain live cell nuclei and the other is cell membrane impermeant and will only fluorescence when cell membranes have been disrupted. Initial imaging and focus of the microscope are accomplished using CY5 LED cube for excitation and emission in order to detect the nuclei of cells stained with the cell permeant stain. With NETosis, cell membranes become disrupted allowing the impermeable dye access to the cytoplasm. Entry of the dye allows an increase in green fluorescence as a result of binding to nuclear DNA. With time, the nuclear membrane breaks down and karyoplasm and cytoplasm contents mix and the nuclear contents are released. The extracellular DNA is also accessible to the green dye, resulting in a greater area of green fluorescence, which is detected with a GFP LED cube.
Image based analysis was used to quantify the degree of NETosis. After background subtraction, the total area of identified green fluorescence in the field of view was used to measure the degree of NETosis. The compounds phorbol 12-myristate 13-acetate (PMA), A23187, and Nigericin were shown to stimulate NETosis in dHL-60 cells in a concentration dependent manner. Several NADPH oxidase inhibitors, including diphenylene iodonium (DPI), VAS 2870, and Apocynin were shown to inhibit the stimulatory effects of PMA, while the Protein Kinase C-α inhibitor Go6976 did not which suggests that PMA acts through a mechanism other than Protein Kinase C. The NETosis stimulation caused by the ionophore compounds, A21387 and Nigericin was not effected by DPI.