Bz x advanced analysis software bz h3ae

EnSCs were treated for 96 hr prior to washing and fixation with 4% paraformaldehyde for 10 min at room temperature and permeabilization with 0.1% Triton X-100 in PBS for 5 min. Cells are washed thrice with PBS then incubated in 3% BSA in PBS for 1 hr at room temperature to block non-specific binding. Then, rhodamine phalloidin (1:50 in blocking solution, Molecular Probes, Eugene, OR) was added for 20 min at room temperature. EnSCs were washed thrice in PBS before nuclei were labelled with DAPI (1:10,000 in PBS) (Life Technologies) and viewed on a Keyence BZ-X700 inverted fluorescence microscope (Keyence, Itasca, IL). Images were captured and merged using BZ-X advanced analysis software (BZ-H3AE, Keyence).

NET release was quantified by measuring dsDNA release into culture supernatants using the Quant-iT PicoGreen dsDNA assay (Thermo Fisher Scientific, Waltham, MA) as previously described (Tong et al. 2019 ). Fluorescence was measured at 485/535nm using the Tecan Infinite M1000 Pro microplate reader (Thermo Fisher Scientific). NET release was also visualized by immunofluorescence. Briefly, extracellular DNA was stained using Sytox Green (167nM, Thermo Fisher Scientific). Cells were then fixed in 4% paraformaldehyde (PFA) before staining with antibodies against citrullinated histone 3 (#ab5103, 1:100 dilution, Abcam, Cambridge, UK). Primary antibody binding was detected using an Alexa Fluor 564-labelled anti-rabbit antibody (1:500 dilution, Thermo Fisher Scientific). Cells were viewed using the Keyence BZ-X700 inverted fluorescence microscope (Keyence, Itasca, IL). Images were captured and merged on the BZ-X advanced analysis software (BZ-H3AE, Keyence).