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Senile plaques are an important histological hallmark of Alzheimer's disease. They mainly consist of the fibrillar peptide β-amyloid (Aβ) and are surrounded by activated microglia and astrocytes. Microglia in the vicinity of senile plaques express high levels of proinflammatory cytokines and neurotoxic substances, which are believed to influence disease progression. One important cytokine in Alzheimer's disease is IL-1β. Stimulation of cultured primary microglia by synthetic fibrillar Aβ causes the release of IL-1β via activation of the NLRP3 inflammasome.Here we provide protocols for the preparation of primary microglial cultures and synthetic oligomeric and fibrillar forms of Aβ.
Tantalum (Ta) is gaining attention as a biomaterial in bone tissue engineering. Although the clinical advantage of Ta-based implants for primary and revision total joint replacement (TJA) has been well documented, few studies investigated the effect of wear products of Ta implants on peri-implant cells, and their potential contribution to aseptic implant loosening. This study is aimed at examining the cytotoxicity, oxidative stress, and proinflammatory potential of Ta and TiO2 nanoparticles (NPs) on macrophages in vitro. NPs were characterized using scanning electron microscopy, dynamic light scattering, and energy-dispersive X-ray. To test the NP-mediated cellular response in macrophages, THP-1-derived macrophages were challenged with both NPs, and cytotoxicity was analyzed using CCK-8 and LDH assays. Flow cytometry was used to investigate particle uptake and their internalization routes. NP-mediated oxidative stress was investigated by measuring the production of reactive oxygen species, and their proinflammatory potential was determined by quantifying the production of TNFα and IL-1β in cell culture supernatants using ELISA. We found that both Ta and TiO2 NPs were taken up through actin-dependent phagocytosis, although TiO2 NPs did also show some involvement of macropinocytosis and clathrin-mediated endocytosis. Ta NPs caused no apparent toxicity, while TiO2 NPs demonstrated significant cytotoxicity at a concentration of over 100μg/mL at 24 h. Ta NPs induced negligible ROS generation and proinflammatory cytokines (TNFα, IL-1β) in macrophages. In contrast, TiO2 NPs markedly induced these effects in a dose-dependent manner. Our findings indicate that Ta NPs are inert, nontoxic, and noninflammatory. Therefore, Ta could be considered an excellent biomaterial in primary and revision joint arthroplasty implants.
MotiQ is an open-source software for automated quantification of microglial motility and morphology. MotiQ can be applied to in vivo, ex vivo, and in vitro data from confocal, epifluorescence, and two-photon microscopy. MotiQ is not limited to microglia—it can also be applied to other cell types.
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