Bz 2 analysis system

The cells transfected with fluorescent probes were cultured in tissue culture-coated glass bottom dishes (Greiner Bio-One). Medium was then replaced with CO₂-independent incubation buffer described above and cells were placed on the microscope stage (BIOREVO BZ-9000 microscope equipped with a CFI Plan Apo VC60xH oil immersion lens) maintained at 37°C in an ambient atmosphere. After the addition of E-IgG, the fluorescent images were collected every 1 min. The intensity of the phagosome-associated fluorescence was analyzed using a BZ-II analysis system (Keyence, Osaka, Japan). In the figures where the time courses of two probes are compared, the fluorescence intensity of each probe was shown as % of respective maximum value. Time zero in the figures indicates the time when the mCherry-Inpp4a fluorescence around E-IgG peaked.

Control-AP (pAPtag-5 vector) and draxin-AP constructs were transfected into HEK293T cells (Riken BRC)12 (link). After 4–5 days, the conditioned media was harvested and then concentrated with the Amicon Ultra-15 filter device (Millipore). In the conditioned media, draxin-AP was detected using western blotting with an anti-draxin antibody (1:1,000)12 (link). Control- and draxin-AP concentrations in the conditioned medium were determined with the SensoLyte pNPP Secreted Alkaline Phosphatase Reporter Gene Assay Kit (AnaSpec). Draxin-AP binding to the dissociated neurons and brain sections was investigated as previously described12 (link)65 (link). We quantified draxin-AP signals on the growth cones of dissociated neurons from the neocortex, anterior dorsal thalamus and posterior dorsal thalamus. For the visualization of draxin-AP binding, neurons were stained with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium for 12 or 24 h at room temperature. We calculated the normalized AP signal intensity, which was the signal intensity at the growth cones of dissociated neurons divided by the signal intensity of the background. In each condition, AP signals were measured for three neurons using the BZ-II analysis system (Keyence) and mean values were calculated. Statistical analyses were performed on the data obtained from three independent experiments.