Hoechst 33342

Sections were dried at room temperature for an hour, rehydrated in PBS, permeabilized with 0.5% Triton X-100 (Sigma) in PBS solution and blocked to saturate non-specific antigen sites using 5% (vol/vol) goat serum-PBS (Jackson Immunoresearch Labs, West Grove, PA) at 4°C overnight. On the next day, the sections were incubated with 0.05% Tween 20 and incubated in anti-Ki67 antibody (Abcam, Cambridge, UK), anti-γH2A.X antibody (Abcam, Cambridge, UK) and propidium iodide (500 nM) (ThermoFisher Scientific, Somerset, NJ) for 4 hours. Later, the sections were washed using PBS-T and incubated with anti-rabbit Alexa flour-647 for 2 hours in the dark. Later, the sections were imaged on incubated with Hoechst 33342 (Abcam, Cambridge, UK). The sections were mounted on slides using fluorescence mounting medium and observed on a fluorescent microscope.

γ-H2AX foci were detected using immunofluorescence as described previously [27 ]. Briefly, cells were seeded into each well of a 6-well plate with a glass coverslip. At harvest, cells were fixed in 4% paraformaldehyde for 10 min, washed three times in Tris-HCl (50 mM, pH 8.0), and incubated for 30 min with blocking buffer (0.02% Triton X-100, 10% horse serum, and 150 mM NaCl in Tris-HCl (50 mM, pH 8.0)). Glass coverslips were then incubated with primary rabbit anti-γ-H2AX polyclonal antibodies (ab11174, Acam, Cambridge, UK; 1:1000 dilution in blocking buffer) at room temperature for 1 h, washed three times in washing buffer (0.02% Triton X-100 and 200 mM NaCl in Tris-HCl (50 mM, pH 8.0)), and incubated with secondary Alexa Fluor 488 goat anti-rabbit IgG antibodies (ab150077, Abcam, Cambridge, UK; 1:300 dilution in blocking buffer) and nuclear counterstaining reagent Hoechst33342 (Abcam, Cambridge, UK) at room temperature for 1 h. Coverslips were mounted with Fluoroshield reagent (ab104135, Abcam, Cambridge, UK). Foci were visualized on a Leica DMI6000 microscope with 100× immersion objective. γ-H2AX foci were counted visually or using ImageJ. More than 100 cells in each experimental group were chosen for γH2AX quantitation.

Cells were fixed with 4% paraformaldehyde in PBS 1X for 15 min and permeabilized with 0.25% Triton-X100 in PBS 1X for 5 min. Then, the cells were incubated with 3% BSA in PBS 1X for 1 h, followed by primary and secondary antibodies in the same solution for 1 h each, at RT in a humid chamber. Finally, the cells were stained with 1 μg/mL Hoechst 33,342 (Abcam). Coverslips were mounted on DAKO Fluorescence Mounting Medium (Agilent Technologies).

CA1a cells were pre‐seeded on poly‐D‐lysine (Gibco, USA) coated 12 mm coverslips (Citoglass, China) 24 h prior to treatment. Following treatment with FAM‐NC‐ASO‐loaded RBCEVs, the coverslips were rinsed with fresh media, and stained with CellMask™ Deep Red Plasma Membrane Stains (Thermo Fisher Scientific) for 10 min at 37°C. Cell were rinsed twice in PBS and stained with Hoechst 33342 (Abcam) for 5 min at room temperature. The coverslips were rinsed three times with PBS before being fixed for 12 min using 4% paraformaldehyde (Alfa Aesar, USA) in PBS. The coverslips were subsequently washed three times with PBS followed by a final wash with distilled water before being mounted on slides using anti‐fade fluorescence mounting medium (Abcam). Images were acquired using an Olympus FV3000 confocal microscope (Olympus, Japan). Image acquisition was conducted using FluoView software while further analysis and quantification was conducted using ImageJ v1.8.0. Cell areas were selected as regions of interest (ROIs) based on the dilated mask of Hoechst signals. FAM signals were measured as mean pixel intensity of ROIs. Total measurement area covered 1200 to 1600 cells in each condition.

Neutrophils on glass coverslips were stained with antibodies of mouse anti-MPO (2C7; Abcam; 1:500), rabbit anti-histone H3 (H3Cit; Abcam;1:250), rabbit anti-elastase (NE; Abcam; 1:1000), and mouse anti-PAD4 (OTI4H5; Abcam; 1:200). We washed the coverslips and incubated the neutrophils with the secondary antibodies, i.e., goat anti-rabbit IgG Alexa 568 (Abcam, 1:200) and goat anti-mouse IgG Alexa 488 (Abcam, 1:500). The nuclei were stained with Hoechst 33,342 (Abcam; 1:500) for 5 min. We mounted the coverslips on slides for analysis under a photographic camera (Olympus Co., St. Laurent, Quebec, Canada) attached to the Olympus BX-51 microscope (Olympus BX51, Olympus Co., Tokyo, Japan). The camera captured the images for digitalization (Oculus TCX, Coreco, Inc., St. Laurent, QC, Canada) and were subsequently processed by the software Image-Pro Plus 6.0. We identified NETs as the area of MPO and H3Cit localization, neutrophil activation NE, and PAD4 expression.