Cellr software

Bright field images were obtained using a Leica DMR microscope (Leica Microsystems CMS GmbH, Wetzlar, Germany) and a Leica DFC 480 camera (Leica Microsystems Ltd, Heerbrugg, Switzerland); images were processed with the Leica Application Suite 2.3.3 software (Leica Microsystems Ltd). Fluorescent images were taken using a Spinning Disc microscope Olympus BX-DSU (Olympus Corporation, Shinjuku-ku, Tokyo, Japan) and acquired with ORCA IR2 Hamamatsu camera (Hamamatsu Photonics, Higashi-ku, Hamamatsu City, Japan). Images acquired using the Olympus Cell^R software (Olympus Soft Imaging Solutions, Munchen, Germany) were processed using the deconvolution software AutoQuantX 2.2.2 (Media Cybernetics, Bethesda, MD, USA) and ImageJ^® software (National Institute of Health, Bethesda, MD, USA).

Fluorescent images were acquired using a Spinning Disc microscope Olympus BX-DSU (Olympus Corporation, Shinjuku-ku, Tokyo, Japan) with ORCA IR2 Hamamatsu camera (Hamamatsu Photonics, Higashi-ku, Hamamatsu City, Japan) and Olympus CellR software (Olympus Soft Imaging Solutions, Munich, Germany) or confocal laser scanning microscope (Nikon C1 Plus; Nikon, Tokyo, Japan). Images were deconvoluted in AutoQuantX 2.2.2 (Media Cybernetics, Bethesda, MD, USA) and processed using FIJI (National Institutes of Health, Bethesda, Maryland, USA; (Schindelin et al., 2012 (link)) and CellProfiler (McQuin et al., 2018 (link)).

Light field photomicrographs were taken using a Leitz-Leica DMRBE microscope (Wetzlar, Germany) equipped with a DFC290 digital camera. Fluorescent images were obtained using an Olympus BX-DSU Spinning Disc microscope (Olympus Corporation, Shinjuku-ku, Tokyo, Japan) equipped with ORCA IR2 Hamamatsu camera (Hamamatsu Photonics, Higashi-ku, Hamamatsu City, Japan) and Olympus Cell-R software (Olympus Soft Imaging Solutions, Munchen, Germany). A stack of 0.5 µm thick was collected. The images were processed using the deconvolution software AutoQuantX 2.2.2 (Media Cybernetics, Bethesda, Maryland, MD, USA) and ImageJ^® software (National Institute of Health, Bethesda, Maryland, MD, USA). Images are shown in Figure 4D–F) were acquired using a Nikon confocal microscope, Eclipse 80i, and analyzed with the EZ-C1 program version 3.90 NIKON.

Neutrophils (1 × 10⁶/mL) were stimulated with PMA or P. aeruginosa PAO1 (MOI 10) in the presence or absence of DPI, seeded (100 µL) into clear 96-well plates and incubated at 37 °C in 5% CO₂. At 30 and 45 min after stimulation, some cells were treated with DPI. Four hours after PMA addition, NETs were stained with Sytox green (200 nM). Cells were viewed on an Olympus IX-81 live cell inverted microscope and images captured using a XM10 monochrome fluorescence charge-coupled device camera and cell˄R software (Olympus Soft Imaging Solutions, Münster, Germany).

Standard preparations stained by Giemsa were inspected under a BX 50 microscope (Olympus, Tokyo, Japan) and images were photographed under immersion objective 100× using a DP 71 CCD camera (Olympus). Pictures from FISH and C-banding were captured by an IX81 microscope (Olympus) equipped with an ORCA-AG CCD camera (Hamamatsu Photonics, Hamamatsu, Japan). The digital images from the FISH and fluorescent banding were pseudocolored (red for Cy3 and CMA₃, green or blue for DAPI) and superimposed with the Cell^R software (Olympus Soft Imaging Solutions GmbH, Muenster, Germany). Fluorescent banding was evaluated under immersion objective 100× using a Provis AX70 Olympus microscope with an appropriate fluorescence filter set. Images were photographed by a black and white DP30W CCD Olympus camera for each fluorescent dye using Olympus Acquisition Software. Karyotypes were arranged in Corel PHOTO-PAINT X4 software (Corel, Ottawa, ON, Canada). Composed images were optimized and arranged using Adobe Photoshop CS6 (Adobe Systems, San Jose, CA, USA).

HUVEC cells were fixed with methanol for 15 minutes at 4 °C, air- dried and blocked with 1% bovine serum albumin in phosphate buffered saline (BSA/PBS) buffer for 30 min. The cells were incubated at 4 °C overnight with the corresponding primary antibodies, washed and labelled with the appropriate fluorophore -conjugated secondary antibodies (Dianova) for 1 hour at room temperature. Nuclei were visualized by counterstaining with DAPI (2 μg/mL, diluted in PBS) for 10 minutes and cells were mounted with Mowiol (Fluka, Missouri, USA). Determination of gelatinase activity was performed by incubating methanol-fixed cells with fluorescein-conjugated DQ-gelatin (EnzChek gelatinase assay kit; ThermoFisher Scientific, Massachusetts, USA) for 1 hour at 37 °C as per manufacturer’s instructions. Immunofluorescence staining intensity was captured and quantified by using the Cell^R software (Olympus, Hamburg, Germany) analyzing at 4–5 different regions of the specimen per experimental group. Exposure times during digital imaging were kept constant. TissueGnostics/TissueQuest microscopy (Axiovert 200 M, Zeiss, Germany) and software were utilized to simultaneously correlate the number of Ki67 positive cells with the Ki67 signal intensity in a scatterplot graph format.

Ex vivo perfused vein segments were fixed in Dent’s fixative (80% methanol, 20% DMSO) for 30 min at room temperature. Vessels were then rehydrated in PBS with decreasing methanol content (75%, 50%, 25%) for 10 min, washed and incubated with a fluorescein-conjugated DQ-gelatin (EnzChek gelatinase assay kit; ThermoFisher Scientific, Massachusetts, USA) for 1 hour at 37 °C. To visualize HO-1 expression, vessels were incubated with a primary anti-HO-1 antibody overnight at 4 °C, washed in PBS and incubated with the corresponding secondary antibody overnight at 4 °C. 4′,6-Diamidin-2-phenylindol (DAPI, 2 μg/mL; Invitrogen, ThermoFisher Scientific, Massachusetts, USA) diluted in PBS was used for visualization of nuclei. Vessels were mounted longitudinally onto glass slides in the presence of Mowiol mounting media (Fluka, Sigma-Aldrich, Missouri, USA). Fluorescence signal intensity was captured and quantified using the Cell^R software (Olympus, Hamburg, Germany).