Ix81 inverted fluorescence microscope

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.

To measure the annealing of actin filaments fluorescence microscopy assays were performed. Actin filaments (10 µM, F-actin) were polymerized for 2 hr at room temperature in 4 mM Tris-HCl (pH 7.0), 0.1 mM CaCl2, 0.2 mM ATP, 0.5 mM DTT, 1 mM EGTA, 1 mM MgCl2 and 50 mM KCl (F-buffer). The F-actin solution was then diluted to 1 µM using F-buffer in the absence or presence of actin-binding proteins (100 nM capping protein or 100 nM dDAAM FH1-FH2 or 1 µM skeletal muscle TM or 100 nM dDAAM FH1-FH2 and 1 µM skeletal muscle TM). The samples were incubated overnight. For investigation of the annealing, Alexa-488-phalloidin labeled samples were sheared five times through a 26 gauge needle. Samples were diluted 100 fold into microscopy buffer (F-buffer supplemented with 50 mMDTT, 5 mM DABCO and 0.5% (w/v) methylcellulose) 0 and 60 minutes after shearing and processed for microscopy observations. Single actin filaments were observed with an Olympus IX81 inverted fluorescence microscope using a 100× objective (NA1.4) and a CCD camera (Orca ERG Hamamatsu). The length of the actin filaments was measured and analyzed with ImageJ. Under each condition 3–4 independent measurements were performed and 300–600 filaments were analyzed. Statistical analysis was carried out using Microsoft Excel or Microcal Origin 6.0.