Poly l lysine coated glass coverslips

VCaP cells were cultured on poly-L-lysine-coated glass coverslips (BD Bioscience) in androgen-deprived medium for 2 days. Cells were induced with 0.1 nmol/L R1881 and cultured for another 48 hours. Cells were fixed with 4% paraformaldehdye buffered in PBS, permeabilized with 0.1% Triton X-100 in PBS, and blocked with 1% normal horse serum (Vector Laboratories) before incubating with purified IgG. Secondary antibody (Alexa Fluor-488 goat anti-human; Invitrogen) was subsequently applied together with DAPI (40, 6-diamidino-2-phenylindole). F-actin was stained with Alexa Fluor-594 phalloidin (Invitrogen). ERG MAb 9FY was used as a positive control and stained with Alexa Fluor-488 goat anti-mouse secondary antibody. Images were captured using a 40x/0.65 N-Plan objective on a Leica DMIRE2 upright microscope with a QImaging Retiga-EX CCD camera controlled by OpenLab software (PerkinElmer), converted into color, and merged by using Adobe Photoshop.

To assess the effects of IFX on NETosis generation, HD neutrophils (n = 3) were isolated and pretreated for 15 min at 4 °C with FCRII blocking Reagent (Miltenyi Biotec, Bergisch Gladbach, Germany). Then, neutrophils were washed with PBS, centrifuged at 300 g for 10 min, seeded in 24 well plates on poly-L lysine-coated glass coverslips (BD Biosciences) (6 × 10⁵ cells per well) in RPMI 1640 supplemented with 20% BSA (PanReacAppliChem, Barcelona, Spain), 2 mM L-glutamine (Biowest, Nuaillé, France) and 1% ZellShield (Minerva Biolabs, BMBH, Berlin, Germany) at 37 °C and 5% CO₂. Next, neutrophils were treated with TNF-α (8 ng/mL; PrepoTech, Rocky Hill, NJ, USA), a potent NETosis inducer, in the presence or in the absence of IFX (100 mg/ml; Pfizer, NY, USA) for 2 h. NETotic response was evaluated as previously described.
To evaluate the effects of IFX on the proinflammatory profile of mononuclear cells promoted by NETs, PBMCs from previous HDs (n = 3) were seeded in 24-well ultra-low attachment plate (Sigma Aldrich) (1 × 10⁶ cells per well) and treated for 24 h with supernatants obtained from above-described in vitro experiments which were pre-incubated in the presence or absence of 15 U/ml DNAse I (NZYTech-Genes & Enzymes, Lisbon, Portugal) for 10 min. PBMCs were harvested for protein and RNA determination.

5×10⁴ isolated PMNs seeded on poly-L-lysine-coated glass coverslips (BD Biosciences) in tissue-culture wells and allowed to settle prior to stimulation as described above. Coverslips were rinsed with ice-cold HBSS and the cells fixed with 4% paraformaldehyde and blocked overnight (HBSS with 10% goat serum, 1% BSA, 0.1% Tween20, and 2 mM EDTA) at 4°C. NETs were detected with rabbit anti-MPO (Dako) and rabbit anti-citrullinated histone H3 (citH3, Abcam). Secondary antibodies were goat anti-rabbit IgG AF555 and goat anti-rabbit IgG AF488 (Invitrogen). DNA was stained with 4',6-diamidino-2-phenylindole (DAPI, Sigma) and NETs were visualized using a Zeiss Axioplan 2 Imaging fluorescence microscope in conjunction with a Zeiss AxioCam MRm monochromatic CCD camera and analyzed with Axiovision 4.8.2 software (Carl Zeiss).