Bz 9000e

Pictures were taken with the light and fluorescence inverted microscope Keyence BZ-9000E, (Keyence Corporation, Higashi-Nakajima, Higashi-Yodogawa-ku, Osaka, 533-8555 1, Japan) using the objectives ×20 (Nicon Plan Apo, 20X/0.75, DIC N2 OFN25 WD 1.0) and ×40 (Nicon Plan Apo, 40X/0.95, DIC M/N2 OFN25 WD 0.14). All pictures were acquired and processed with the Keyence camera and software. The camera is installed in the Keyence BZ-9000E microscope (2/3 inch, 1.5 million pixel monochromer CCD Fotosensor (with LC Filter)) with shooting condition 1360 × 1024 pixel. The acquisition software is delivered with the Keyence BZ-9000E microscope: Program “BZ-II Viewer” (Version: 2.1.00a0.0100.0101.0100.0003). Image acquisition was performed in the following conditions: detector gain +12 dB, bit depth 24, fluorescence filters OP-66834 BZ Filter DAPI-BP (excitation wavelength 360/40 nm, absorption wavelength 460/50 nm), OP-66836 BZ Filter GFP-BP (excitation wavelength 470/40 nm, cold mirror wavelength 505 nm, absorption wavelength 535/50 nm), OP-66838 BZ Filter TexasRed (excitation wavelength 560/40 nm, absorption wavelength 630/60 nm), excitation time 1/15–1/90 s; multifluorescence image acquisitions were performed successively. The obtained pictures were analyzed using the “BZ-II Analyser” (Version 2.1) software, which is delivered with the Keyence BZ-9000E microscope.

To assess the effects of OPN on NSC numbers, recombinant rat osteopontin (OPN; R&D Systems) with concentrations ranging from 1.25–12.5 μg/ml was added to the cultures 1 h after re-plating. To assess the role of CXCR4 in mediating any effects of OPN on cell numbers, we blocked this receptor with the CXCR4-antagonist AMD3100 (Tocris Bioscience, Bristol, UK) at a concentration of 10 μM in some wells directly after re-plating, resulting in a pre-incubation time of NSC with AMD3100 of 1 h before adding OPN. After 72 h, dead cells were stained with propidium iodide (Life Technologies). All cells, irrespective of viability, were counterstained with Hoechst 33342 (Life Technologies) and representative pictures were taken using an inverted fluorescence phase-contrast microscope (Keyence BZ-9000E; Keyence, Neu-Isenburg, Germany). Ten images were taken per well of a 24-well plate, and both Hoechst-stained and propidium iodide-stained NSC were counted manually. To calculate the ratio of living cells, the number of propidium iodide-negative cells was divided by the total (Hoechst-stained) number of NSC in each sample, and mean values were established among equally treated samples. Results were expressed as percent of the control ± standard error of the mean (SEM).

Fluorescence microscopy and scanning electron microscopy was used for visualization of NETs. For FM, 10⁶ neutrophils per milliter were incubated in iIC-coated ibiTreat μ-slides (ibidi) for 7 h when iIC was used as stimulus or for 4 h in Poly-Ly-Lysin coated μ-slides when PMA was used as stimulus. Following fixation with 4% paraformaldehyde (Sigma-Aldrich), staining of MPO and DNA by using mouse anti-human MPO antibody (1:500, AbD Serotec, Germany) and SYTOXgreen was carried out as described previously (4 (link), 56 (link)). Samples were analyzed with the AxioVert A.1 using the the Axiocam HRc and Axio Vision Rel. 4.8 software (all Carl Zeiss, Germany) or with the Keyence BZ-9000E using the BZ II Analyzer Software (KEYENCE, Germany).
For scanning electron microscopy, neutrophils (10⁶ per milliliter) were settled on iIC-coated or uncoated (medium control, PMA) thermanox coverslips (Greiner BioOne). Following incubation for 7 h (iIC)/4 h (PMA), the supernatant was removed and samples were fixed with 1 ml Monti-Graziadei solution and processed for SM as described (56 (link)). Preparates were examined with a Zeiss EVO HD 15 (Zeiss).