Astrocyte-Neuron Differentiation Assay

DITNC1 astrocytes were seeded at a density of 5 × 10⁵ cells/cm² and incubated to 90% confluency in 6-well glass bottom plates. Then, the cells were treated with OMVs (2.5 μg/ml) or TNF (10 ng/ml) for 12 h or 48 h, respectively. Next, the medium was eliminated, and cells were washed twice with PBS and fixed with 4% paraformaldehyde for 15 min. The reaction was then stopped with 0.1 mM glycine. Following extensive washing with PBS, CAD cells labeled with Cell Tracker Green CMFDA (10 µM) were seeded over fixed astrocytes at a density of 1 × 10⁴ cell/cm² in DMEM F12 media, supplemented with 8% FBS and antibiotics [55 (link)]. Before 24 h of culture, the media was replaced with fresh SFM containing 50 ng/ml sodium selenite (S5261, Sigma-Aldrich) to induce morphologically visible neuronal differentiation. After 24 h, neuronal processes were captured with an Epifluorescent Spinning disk microscope (Olympus). The neurite extension length was determined using the NeuronJ plug-in of the v1.8 NIH ImageJ Software, as previously described [56 (link), 57 (link)].