Ab25989

All reagents were purchased from Sigma (St. Louis, MO) unless indicated otherwise. Dyes and secondary antibodies were obtained from Thermo-Fisher (Waltham, MA). RNAscope 2.5 HD Detection of s-sense COVID-19 for RNA detection was used (Hayward, CA). Antibodies for macrophages (Iba-1, Ab5076), lymphocytes (CD3, ab11089), endothelial cells (Von Willebrand factor, ab194405), epithelial cells (EpCam, ab7504), myeloperoxidase (MPO, ab25989), CD8 (CD8, ab22378), CD20 (B cells, ab9475), and smooth muscle actin (SMA, ab21027) were obtained from Abcam, MA. Vimentin (sc-52721) from Santa Cruz (Santa Cruz, CA) and the antibody for SARS protein M (APO90991su-n) were obtained from Origene (Rockville, MD). All experiments were performed under the University of Texas Medical Branch (UTMB) and the NIH regulations.

Cells at a concentration of 1 × 10⁶ cells per ml were plated on a 96-well plate, and incubated with inhibitors for 1 h at 37 °C. Following induction of NETosis, reaction proceeded for an allotted amount of time at 37 °C before being terminated with 4% (w/v) paraformaldehyde (Sigma Aldrich) overnight. Cells were permeabilised with 0.1% Triton X-100 for 15 min and then blocked with 2.5% (w/v) bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 1 h. MPO was probed for using mouse anti-myeloperoxidase antibody (ab25989, Abcam) at a 1:500 dilution. Cleaved-caspase 3 was probed by rabbit anti-cleaved-caspase 3 antibody (ASP175, Cell Signalling) at a 1:500 dilution. Citrullinated histone was probed by rabbit anti-histone H3 (citrulline R2+R8+R17) antibody (ab5103, Abcam) at a 1:500 dilution. DAPI (10 μM; ThermoFisher Scientific) at 1:333 dilution was used for visualising DNA. Imaging was done using Olympus IX81 inverted fluorescence microscope with a Hamamatsu C9100-13 back-thinned EM-CCD camera and Yokogawa CSU ×1 spinning disk confocal scan head.

Serially cut frozen slides were air-dried and fixed in prechilled 100% acetone). Slides were rinsed in distilled water and then washed in TBS-T. Endogenous peroxidase activity was quenched via incubation with 5% (v/v) H₂O₂ (Sigma-Aldrich, H3410) in dH₂O. After 30 minutes, the slides were washed and incubated in serum-free protein block (DAKO, X0909) for 30 minutes, after which excess liquid was removed. Serial sections were then incubated with 1:1000 (v/v) anti-MPO (mouse, monoclonal; Abcam, ab25989) or 1:1000 (v/v) anti-NE (rabbit, monoclonal; Abcam, ab131260) or 1:500 (v/v) anti-CitH3 (rabbit, monoclonal; Abcam, ab219407) for 1 hour in antibody diluent (bovine serum albumin [1% w/v], Sigma-Aldrich, A7906) and Triton-X100 (0.5% v/v, Sigma-Aldrich, 270733) in TBS-T. Slides were further washed in TBS-T and incubated with horse radish peroxidase (HRP)-coupled secondary antibody (anti-mouse, DAKO, K4001; anti-rabbit, K4003) for 30 minutes. Following washing in TBS-T, slides were incubated for 10 minutes with Opal690 fluorophore (PerkinElmer, FP1497) in tyramide signal amplification (TSA) buffer (PerkinElmer, FP1498, 1:50 v/v dilution in 1x plus amplification buffer) and subsequently washed. Specimens were incubated in a 1:800 (v/v) dilution of Spectral DAPI for 10 minutes, washed, and then cover-slipped with fluorescence mounting medium.

Human neutrophils were isolated, seeded (200,000/well) in 24-well plates on glass coverslips and activated to undergo NET formation as described above. After fixation with 2 % PFA (Roth) over night, cells were permeabilized 0.1 % TritonX (Merck) and incubated with 5 % FCS (Biochrom) to block unspecific antibody binding. Subsequently, cells were stained using monoclonal anti-human MPO (IgG, mouse) as primary antibody (Abcam, ab25989, 1:500) and polyclonal anti-mouse Alexa 555 (IgG, goat) as secondary antibody (Life technologies, A21422, 1:2000). Neutrophil DNA was stained with 1.62 μM Hoechst (Sigma-Aldrich) as described above. After the staining procedure, cells were stored protected from light at 4°C. Representative confocal fluorescence images were obtained with the olympus IX83 inverted microscope (software: Olympus Fluoview Ver.4.2, Olympus) and recorded 60x magnified (UPlanSApo 1.35 oil, Olympus). All pictures were recorded at equal exposure times for MPO to ensure comparability.

As previously described by Vong et al. [29 (link)], human neutrophils were adhered onto glass coverslips in 24-well non-treated culture plates by incubation for 30 min at 37 °C. They were then inoculated with T. rubrum conidia or hyphae and incubated for an additional 3 h at 37 °C. For the positive control, 100 nM phorbol myristate acetate (PMA) was added to stimulate neutrophils. Monolayers were fixed with 4% paraformaldehyde (Labsynth, Diadema, Brazil) in PBS (Sigma-Aldrich, Burlington, VT, USA), permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, Burlington, VT, USA), and blocked with 3% albumin bovine serum (BSA), (Sigma-Aldrich, Burlington, VT, USA). Samples were incubated with primary antibodies: mouse monoclonal anti-myeloperoxidase (AB25989, Abcam, Cambridge, UK) and rabbit polyclonal anti-histone H3 (AB5103, Abcam, Cambridge, UK), followed by exposure to a secondary antibody conjugated to Alexa Fluor 488 (ab150113, Abcam, Cambridge, UK) or Alexa Fluor 568 (ab175471, Abcam, Cambridge, UK), and nuclear staining was made with Hoechst dye. Cells were analyzed by fluorescence microscopy in a Nikon eclipse 80i.

Cells and NETs were fixed with paraformaldehyde (4% (w/v) for 10 min; 2% (w/v) for overnight), and immunostained with various NET markers. Mouse anti-myeloperoxidase antibody (ab25989, Abcam) at 1:500 dilution was used for staining MPO (with secondary antibody conjugated with a green fluorescence Alexa fluor 488 dye; 1:2000 dilution; ThermoFisher Scientific), while rabbit anti-citrullinated histone 3 antibody (ab5103, Abcam) at 1:500 dilution was used for detecting the presence of CitH3 (with secondary antibody conjugated with a far red fluorescence dye Alexa fluor 647; 1:1000 dilution; ThermoFisher Scientific). DNA was stained with DAPI (1:1000 dilution). To obtain high-resolution images, 8-well chamber slides (Falcon culture slides) were used. After completing the immunostaining, the slides were mounted with anti-fade fluorescent mounting medium (Dako) and glass cover slips (Fisher Scientific). True NETosis was confirmed by MPO colocalization to NET DNA by immunofluorescence confocal microscopy (Olympus IX81 inverted fluorescence microscope equipped with a Hamamatsu C9100-13 back-thinned EM-CCD camera and Yokogawa CSU × 1 spinning disk confocal scan head). The confocal images were taken at 40× magnification with 1.35× objective, and processed by Volocity software (version 6.3, Cell Imaging Perkin-Elmer).