Zen 3.4 blue edition software

The isolated PMNs from each subject were placed on poly-L-lysine pre-coated cover glasses in a 24-well plate with a density of 45 cells per well, incubated at 37 °C with an atmosphere of 5% of CO₂ for 20 min, and let to adhere. Subsequently, cells and the spontaneous release of NETs were fixed with a 4% p-formaldehyde solution for 10 min at RT and washed twice with 0.1 M PBS. The samples were incubated with constant shaking with a blocking solution (5%-BSA, 0.1% Triton 100X, and 0.1 M PBS) for 2 h at RT °C.
For the visualization of in vitro NET release, the samples were incubated overnight at 4 °C with constant shaking with a rabbit anti-human anti-neutrophil elastase antibody (NE) (1:100). Negative controls were performed, leaving out the primary antibody. After washing three times with 0.5 mL of PBS 0.05% tween 20 (PBS-T), the samples were incubated for 2 h with an Alexa 488 goat anti-rabbit IgG antibody (1:800) at RT °C. The samples were rinsed twice with PBS-T, and DNA visualization was performed with 25 μg/mL of propidium iodide (PI). Finally, the specimens were sealed, and the images were acquired with an ApoTome II microscope using ZEN 3.4 (blue edition) software from Carl Zeiss (Jena, Germany).

Oxidative stress in HepG2 cells was determined using a DCFDA/H2DCFDA-Cellular Reactive Oxygen Species (ROS) Assay Kit and fluorescence microscopy. HepG2 cells (1 × 10⁵ cells on chamber slides in 24-well culture plates) were treated with 337 µgmL^-1 of the antitumor peptide of interest, 50 µM of tert-butyl hydrogen peroxide (tbHP; positive oxidant control), 1% DMSO (diluent control), and medium alone (negative control) for 24 h. The generation of ROS in the treated HepG2 cells was then determined using the DCFDA/H2DCFDA-Cellular ROS Assay Kit (Abcam, Cambridge, MA, USA). The treated cells were washed with 1× buffer, stained with 2’, 7’-dichlorofluorescin diacetate (DCFDA) solution (1× buffer containing 10 µM of DCFDA), and incubated at 37 °C for 45 min in darkness, as per the manufacturer’s instructions. After incubation, the stained cells were washed twice with 1× buffer, fixed with cold methanol at room temperature for 20 min, and placed on a glass slide containing 8 µL of mounting medium. Cellular ROS production was observed under confocal microscopy (Nikon Instruments Inc., Melville, NY, USA) at 400× magnification with a filter set appropriate for fluorescein (FITC). Intensity mean value of FITC signal was analyzed using Zen 3.4 (blue edition) software (Zeiss, Oberkochen, Germany).

Photographs of conidia were taken on a Zeiss Primo Star (Carl Zeiss^®, Jena, Germany.) brightfield microscope with a Primo Plan-ACHROMAT 40×/0.65 lens using the AxioCam ICc 1 camera and ZEN 3.4 Blue edition software from Carl Zeiss (Jena, Germany) for the corresponding hyphal measurements, adjusting the scale with a micrometer. Photographs of the root colonized with M. guizhouense strain HA11-2-GFP were taken using a Zeiss Axio Zoom V.16 fluorescence stereomicroscope with Plan Neo Fluar Z 1×/0.25 FWD 56 mm lens 40× zoom.