The tissue Regorafenib clinical trial was further homogenized by filtration (180 μm), trituration and consecutive incubation for 30 min with 1 mg/mL collagenase/dispase (Roche, Germany). The cell suspension was layered
onto a two-level percoll gradient with ρ = 1.08 and ρ = 1.04 g/mL. Mixed brain cells were collected from the lower interface of the gradient and were washed and seeded in Dulbecco’s modified eagle’s medium, supplemented with 10% fetal calf serum and antibiotics. Microglia were collected after 7 days by gently washing the confluent cell layer and collecting the loosely adherent cells. Finally, the microglia were plated in RPMI medium supplemented with 10% fetal calf serum and antibiotics at a density of 0.8 × 106/mL in 96-well plates. After seven days in vitro, macrophages were detached with Accutase®
(PAA, Germany) supplemented with 2 mmol EDTA for 45 min at 37 °C and fixed with 2% paraformaldehyde on poly-l-lysine-coated slides for 60 min at room temperature. Subsequently, the cells were permeabilized and blocked in PBS with 1% bovine serum albumin (BSA)/5% goat serum/0.2% Triton-X-100 for 1 h at room temperature. Labeling with mouse anti-human iNOS monoclonal antibody (R&D Systems, PARP inhibitor USA) was performed at a concentration of 20 μg/ml for 80 min at room temperature followed by staining with secondary antibody AF488 goat anti-mouse (Invitrogen, Germany) for 1 h at room temperature. Slides were mounted with Roti®-Mount FluorCare DAPI (Roth, Germany), and images were acquired on a Nikon eclipse 80i microscope equipped with NIS-elements BR 3.1 software. Aβ(1–40), Aβ(1–42), Aβ(2–40), Aβ(2–42), Aβ(3p–42) and Aβ(5–42) (all Anaspec, USA) were reconstituted Atorvastatin in 1% NH4OH, diluted with H2Odd to reach a final concentration of 1 mg/ml in H2Odd/0.08% NH4OH and stored in aliquots at −20 °C. Yellowgreen
Flouresbrite® (Polysciences, Germany) polystyrene particles (PSP) with a diameter of 1 μm were resuspended at 4.55 × 1010 particles/ml in the respective Aβ-peptide solution for 12 h at 37 °C. After washing, the particles were centrifuged at 10,000g for 10 min and suspended in PBS. For the phagocytosis assay, the particles were diluted in the appropriate cell culture medium to reach a final concentration of 1.5 × 108 particles/ml. The coating of PSP with bovine serum albumin (BSA, Sigma, Germany) was performed equivalently. The AF488-labeled E. coli BioParticles® (Invitrogen, Germany) were reconstituted at 20 mg/ml in H2Odd with 2 mM sodium azide and coated with the respective Aβ-peptides, BSA or opsonizing reagent (OpsR, Invitrogen, Germany) as described above. The E. coli were diluted in cell culture medium to reach a final concentration of 0.8 × 108 particles/ml.pHrodo Green-labeled E. coli BioParticles (Invitrogen, Germany) were reconstituted at 2 mg/mL in PBS and were treated equivalently. The amount of Aβ-peptide bound to the polysterene particles was assessed by staining with Aβ-peptide-specific antibodies and measurement by flow cytometry.
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