Mouse neurotrophin 3 elisa kit

Transfection: Whole inner ears were dissected from C57Bl/6 mice and utricles were removed. The utricles were placed on a 0.45-µm Millicell-HA culture insert (cat# PIHA01250) in a 24-well plate with media (DMEM, N1 supplement, penicillin, neomycin 10⁻³). This process was repeated for each subsequent dissection. The tissues were incubated for 48 h at 37°C/5% CO₂ to induce injury. The vector was added to fresh media (1 µl vector/400 µl medium) into a new 24-well plate and returned to the incubator. The medium was collected and replaced every 72 h ending on the 27th day of collection. The media were then placed in −20°C freezer until further analysis. Analysis was performed in triplicate with five utricles per membrane.
Enzyme-linked immunosorbent assay (ELISA): ELISAs specific for BDNF and NT-3 were used to detect expression levels in the supernatants. Explant supernatant was thawed, and reactivity was tested for the neurotrophins (BDNF: PicoKine^™ ELISA Kit, Boster Biological Technology #EK0307/9; NT-3: mouse Neurotrophin-3 ELISA Kit, Abcam, ab213882).
A subset of neomycin medium collected from neomycin-treated explants which were incubated with Ad28.lat.bdnf were microdialyzed using Pall Nanosep^® Filters (Pall Corp, Westborough, MA United States) per manufacturer’s instructions and subsequently assayed for BDNF content as outlined above.

For the detection of NT-3 in the supernatant of wildtype and Vav3^–/– astrocytes, serum-free neuronal medium was conditioned for 24 h, as previously mentioned under 2.5. Then, the Mouse Neurotrophin-3 ELISA Kit (ab213882) from Abcam was used without any changes of the manufacturers protocol.