Anti neutrophil elastase antibody ab21595

In all, 500 000 human neutrophils were plated on 0.01% poly‐L lysine (Sigma‐Aldrich) coated coverslips (Chemglass Life Sciences LLC) in 24‐well cell culture plates, then incubated with 0.5 mL MSC CM for 3 hours. Studies were done using neutrophils obtained from three unrelated, healthy donors. IRB approval was obtained for collection of human blood samples with signed informed consent. After washing off the CM, S. aureus was added at an MOI of 1 for the indicated time points in HBSS containing calcium, magnesium and 10% autologous human serum. Staining for neutrophil extracellular trap (NET) formation was performed according to a published protocol.28, 30 Cells were immunostained with antibodies for detection of histone H3 using Anti Histone H3 (RM188) Antibody (Caymen Chemical, Ann Arbor, Michigan) and neutrophil elastase Anti‐Neutrophil Elastase antibody (ab21595) (Abcam) with slight modifications for staining in a 24‐well plate. Images were taken on Olympus IX83 spinning disk confocal microscope, by imaging 15 random fields per condition. The NET area was calculated using ImageJ31; and the NET area (in pixels) was determined by merging channel 2 and 3 pixels (representing histone H3 and neutrophil elastase staining, respectively) then normalized to DAPI channel area.

A total of 2 × 10⁵ PMNs were plated in a 24-well plate on poly-L-lysine coverslips and allowed to equilibrate/adhere for 10 min. Subsequently, the cells were stimulated for 3 h with 200 nM PMA and 100 μg/mL MSU to induce NET formation. Next, the cells were fixed with 10% neutral buffered formalin (#HT501128-4L, Sigma-Aldrich) overnight. The following day, the coverslips were blocked and permeabilized with 5% goat serum and 0.1% Triton X-100 for 1 h. Anti-neutrophil elastase antibody (#ab21595, dilution ratio of 1:200, Abcam Inc., Cambridge, UK) was used to visualize NETs. DNA was then stained with 1 μg/mL 4',6-diamidino-2-phenylindole (DAPI; #D1306, Molecular Probes, Eugene, OR, USA). Goat anti-rabbit Alexa 594 (#R37117, Thermo Fisher Scientific) served as the secondary antibody. Images were captured with the help of a laser scanning confocal fluorescence microscope (Olympus Fluoview FV10i) using a 60 × objective.