Csu 1 spinning disk confocal scan head

Cells that had been induced to produce NETs were fixed with paraformaldehyde (4% (w/v) for 10 min; 2% (w/v) onto 8-chamber slides (BD Falcon, New York, NY, USA) overnight, and immunostained with several NET markers. For MPO staining, mouse anti-myeloperoxidase antibody (ab25989, Abcam, Cambridge, MA, USA) at 1:500 dilution was used (with secondary antibody conjugated with a green fluorescence Alexafluor 488 dye; 1:2000 dilution; Thermo Fisher Scientific, Waltham, MA USA)) and DAPI (1:1000 dilution) was used to stain DNA. Eight-well chamber slides (Falcon culture slides) were used to obtain high-resolution images. Following immunostaining, the slides were mounted with anti-fade fluorescent mounting medium (Dako, Santa Clara, CA, USA) and glass cover slips (Fisher Scientific, Markham, ON, Canada)). NETosis was identified by MPO to NET DNA colocalization by an immunofluorescence confocal microscopy (Olympus IX81 inverted fluorescence microscope equipped with a Hamamatsu C9100-13 back-thinned EM-CCD camera and Yokogawa CSU × 1 spinning disk confocal scan head). The confocal images were taken at 40× magnification with 1.35× objective, and processed by Volocity software (Version 6.3, Cell Imaging Perkin-Elmer, Quorum Technologies Inc., Puslinch, ON, Canada).

Cells at a concentration of 1 × 10⁶ cells per ml were plated on a 96-well plate, and incubated with inhibitors for 1 h at 37 °C. Following induction of NETosis, reaction proceeded for an allotted amount of time at 37 °C before being terminated with 4% (w/v) paraformaldehyde (Sigma Aldrich) overnight. Cells were permeabilised with 0.1% Triton X-100 for 15 min and then blocked with 2.5% (w/v) bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 1 h. MPO was probed for using mouse anti-myeloperoxidase antibody (ab25989, Abcam) at a 1:500 dilution. Cleaved-caspase 3 was probed by rabbit anti-cleaved-caspase 3 antibody (ASP175, Cell Signalling) at a 1:500 dilution. Citrullinated histone was probed by rabbit anti-histone H3 (citrulline R2+R8+R17) antibody (ab5103, Abcam) at a 1:500 dilution. DAPI (10 μM; ThermoFisher Scientific) at 1:333 dilution was used for visualising DNA. Imaging was done using Olympus IX81 inverted fluorescence microscope with a Hamamatsu C9100-13 back-thinned EM-CCD camera and Yokogawa CSU ×1 spinning disk confocal scan head.

Cells and NETs were fixed with paraformaldehyde (4% (w/v) for 10 min; 2% (w/v) for overnight), and immunostained with various NET markers. Mouse anti-myeloperoxidase antibody (ab25989, Abcam) at 1:500 dilution was used for staining MPO (with secondary antibody conjugated with a green fluorescence Alexa fluor 488 dye; 1:2000 dilution; ThermoFisher Scientific), while rabbit anti-citrullinated histone 3 antibody (ab5103, Abcam) at 1:500 dilution was used for detecting the presence of CitH3 (with secondary antibody conjugated with a far red fluorescence dye Alexa fluor 647; 1:1000 dilution; ThermoFisher Scientific). DNA was stained with DAPI (1:1000 dilution). To obtain high-resolution images, 8-well chamber slides (Falcon culture slides) were used. After completing the immunostaining, the slides were mounted with anti-fade fluorescent mounting medium (Dako) and glass cover slips (Fisher Scientific). True NETosis was confirmed by MPO colocalization to NET DNA by immunofluorescence confocal microscopy (Olympus IX81 inverted fluorescence microscope equipped with a Hamamatsu C9100-13 back-thinned EM-CCD camera and Yokogawa CSU × 1 spinning disk confocal scan head). The confocal images were taken at 40× magnification with 1.35× objective, and processed by Volocity software (version 6.3, Cell Imaging Perkin-Elmer).