Rabbit anti mpo

Total protein was isolated with NucleoSpin TriPrep kit (Macherey-Nagel) from 3 × 10⁶ PMNs. Proteins from the nuclear and cytoplasmic fractions were isolated by using the Nuclear and Cytoplasmic Protein Extraction Kit (Thermo Scientific). Western blotting was performed by using AnykD Mini-PROTEAN TGX Gels (Biorad, Hercules, CA, USA) and nylon/nitrocellulose membranes (Biorad). Primary and secondary antibodies used were: rabbit anti-PAD4 (Abcam), rabbit anti-MPO (Cell Signalling Technologies, Beverly, MA, USA), mouse anti-β-Actin (Sigma-Aldrich), goat anti-Mouse and/or anti-Rabbit, human anti-HRP (Southern Biotech). HRP activity was detected by using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). Equal loading was verified by using beta-actin or histone H3, when appropriate. Western blots of citrullinated H3 (citH3) protein were performed according to Shechter et al.[25 (link)]. Densitometric analysis and protein quantification of the Western blots was performed by using the ImageJ software.

A total of 1 × 10⁴ or 2 × 10⁵ A549 cells were seeded in 4-well chamber slides or on coverslips in 6-well plates and incubated for up to 24 h at 37 °C. Cells were serum starved for 2–4 h followed by MPO incubation for the indicated timepoints. To investigate MPO uptake by A549 cells, the cells were incubated with heparin (150 µg/mL) for 1 h before adding MPO (10 µg/mL) for 2 h. Cells treated with ddH₂O or DMSO (0.2%) were used as the controls. Cells were rinsed with PBS (3 × 2 min) before fixing and permeabilizing with ice-cold methanol for 20 min at −20 °C. Cells were washed with PBS (3 × 2 min on shaker) and incubated with blocking solution (5% goat serum + 5% BSA in PBS with 0.1% TritonX) for 60 min at room temperature (on shaker). The primary antibody (rabbit anti-MPO 1:500; Cell Signalling, Danvers, MA, USA) was prepared in an antibody diluent (1:10 blocking solution in PBS-TritonX) and incubated overnight at 4 °C. After washing the cells with PBS (3 × 2 min on shaker), they were incubated with the secondary antibody (goat anti-rabbit AF488, 1:500) for 1 h at room temperature in the dark. After washing the cells with PBS (5 × 2 min on shaker), they were mounted using VECTASHIELD with DAPI. The cells were visualized using an Olympus iX70 fluorescence microscope and the Olympus cellSens Dimension 2.3 imaging software with Z-stacking to allow imaging of different cell layers.

Total protein was isolated by NucleoSpin TriPrep kit (Macherey-Nagel) from 5 × 10⁶ neutrophils. All protein concentrations were determined with the MN Protein Quantification Assay (Macherey-Nagel). Western blotting was performed utilizing AnykD Mini-PROTEAN TGX Gels (Biorad) and nylon/nitrocellulose membranes (Biorad). Primary and secondary antibodies utilized were rabbit anti-MPO (Cell Signalling Technologies), rabbit anti-PAD4 (Abcam), rabbit anti-citH3 (Abcam), mouse anti-β-Actin (Sigma), anti-rabbit HRP (Santa Cruz), and anti-mouse HRP (Santa Cruz). HRP activity was detected by using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). Equal loading was verified using beta-actin. Western blots of citrullinated H3 (citH3) protein were prepared as described previously (34 (link)). Gel documentation, densitometric analysis, and protein quantification of the western blots was performed using the ChemiDoc XRS+ imaging system (Biorad) with the ImageLab 4.1 image analysis software (Biorad).