C9100 13 back thinned em ccd camera

For imaging, cells used for the plate reader assay were fixed with PFA (4%, w/v) in PBS buffer for 10 min and permeablized with Triton X-100 (0.1%, w/v). Mouse-α-myeloperoxidase antibody (ab25989, Abcam, Ontario, Canada) at 1 : 500 dilution and an anti-mouse IgG secondary antibody conjugated with Alexa Fluor 555 (Invitrogen) were used for staining MPO. DNA was already stained with Sytox Green for the plate reader assay. The confocal images were taken using the Olympus IX81 inverted fluorescence microscope equipped with a Hamamatsu C9100-13 back-thinned EM-CCD camera and Yokogawa CSU X1 spinning disk confocal scan head (with Spectral Aurora Borealis upgrade) and 4 separate diode-pumped solid state laser lines (Spectral Applied Research, 405 nm, 491 nm, 561 nm, and 642 nm). The objectives used were 20 ×/ 0.75 or 60 ×/ 1.35. The microscope was operated with Volocity software (Perkin Elmer, Waltham, MA). Images taken on the spinning disk confocal microscope were deconvolved by iterative restoration with confidence limit set to 95% and iteration limit set to 20. All images were deconvolved to the confidence limit before reaching iteration limit.

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).

Images were taken on an Olympus IX81 inverted fluorescence microscope using a 60×/1.35 oil immersion objective equipped with a Hamamatsu C9100-13 back-thinned EM-CCD camera and Yokogawa CSU X1 spinning-disk confocal scan head (with Spectral Aurora Borealis upgrade). The unit is equipped with four separate diode-pumped solid state laser lines (Spectral Applied Research, 405, 491, 561, and 642 nm) with emission filters: 447 ± 60, 525 ± 50, 593 ± 40, 620 ± 60, 676 ± 29, and 700 nm ± 75, and 1.5× magnification lens (Spectral Applied Research). Confocal images were taken with an Improvision Piezo Focus Drive. Z-stacks were taken at 0.25 μm. Images taken using the spinning-disk confocal microscope were deconvolved by iterative restoration using Volocity Software (PerkinElmer, Waltham, MA, USA) with confidence limit set to 95% and iteration limit set to 20.

Cells (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 (PFA; Sigma-Aldrich) overnight. Cells were permeabilized with 1% Triton X-100 for 25 min and then blocked with 2.5% (w/v) BSA in PBS for 1 h. PCNA was probed for using mouse anti-PCNA antibody (F-2, Santa Cruz) at a 1:250 dilution. 8-oxoGuanine was probed using for mouse anti-8-Oxoguanineantibody (MAB3560, Millipore Sigma) at a 1:250 dilution. DAPI (10 μM; Thermo Fisher 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.

Fluorescent images were acquired with an Olympus IX81 inverted fluorescence microscope equipped with a 60× objective (1.35 NA), Hamamatsu C9100-13 back-thinned EM-CCD camera, and Yokogawa CSU X1 spinning disk confocal scan head (with Spectral Aurora Borealis upgrade). Images were acquired of multiple z-slices (0.3 µm) and collapsed xy projections presented. Image analysis was performed using Perkin Elmer Volocity software. Single cells were selected and the Pearson's Correlation calculated for the whole cell using Volocity software.

Cells (5 × 10⁶ cells per ml) were plated on a 96-well plate, and incubated with PMA or LPS for 2 h at 37 °C. The reactions were terminated with 4% (w/v) PFA (Sigma-Aldrich) overnight. Cells were permeabilized with 1% Triton X-100 for 25 min and then blocked with 2.5% (w/v) BSA in PBS for 1 h. 8-oxoG was probed for using mouse anti-8-Oxoguanineantibody (MAB3560, Millipore Sigma) at a 1:250 dilution. Plate was 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. Fluorescence was measured using POLARstar OMEGA fluorescence plate reader (BMG Labtech; excitation = 485 nm, emission = 525 nm).