4.5. Quantification of NETs by Fluorescence Spectrophotometry

Quantification of NETs by Fluorescence Spectrophotometry:

Extracellular DNA was stained with 5 µM SYTOX green (Invitrogen, Carlsbad, CA, USA), a fluorescent membrane-impermeable DNA dye. Fluorescence was quantified using a microplate reader at 485/535 nm.

1) Seed the cells at the proper density (e.g. 50 000/ml) in HBSS (+) medium in 96-well plate (non-coated) 
2) Stimulatate cells (e.g. with LPS)
3) At the end of the incubation period prepare SYTOX green solution (Add 1µl 5 mM stock solution to the 999 µl HBSS (+), 1:1000 ratio, 5µM = working solution) and vortex. 
4) Add 10 µl of the Sytox green working solution to each well 
5) Tap the plate on each side to distribute the SYTOX evenly in each well
6) Measure the plate in a microplate reader at 485/535 nm (e.g. Tecan, Infinity F200 Pro)