Prussian blue iron assay kit

At day 29, confluent neuronal cultures were incubated with FeraTrack MRI SPIO nanoparticles (Miltenyi Biotec) overnight in 12-well plates. Cell viability versus SPIO concentration analysis was performed after 24 h incubation. ~100 µg Fe / 1 x 10 6 cells (40 µl Miltenyi Biotec FeraTrack SPIO / 2 ml culture medium) was chosen according to cell viability and manufacturer's instructions. SPIO uptake was confirmed using a Prussian blue iron assay kit (Millipore Sigma) according to manufacturer's instructions. Day 30 SPIO-labeled cultures were encapsulated in neural ribbons plus Fluorescein-Dextran (500 kDa, 1:30 in alginate) and shipped on ice overnight as described above. Collagen phantom slabs were generated from 5 mg/ml PureCol EZ gel Type 1 Collagen solution (Advanced Biomatrix #5074) at RT by plating the solution in 3 cm dish. 60 µm diameter neural ribbons were manually severed to single 3 -4 mm segments containing ~5,000 cells. Single ribbon segments were individually drawn into a 26G custom made curved needle Hamilton syringe with a ~45 degree bend, ~2 mm proximal to the bevel and injected by hand into collagen phantoms. Fluorescein-Dextran in neural ribbon segments was visualized using a Leica M165 FC stereo microscope.