Fig. 4

Internalization profile of osteoclast-derived EV by dorsal root ganglia (DRG) neurons in compartmentalized microfluidic chips. A Schematic representation of the experimental setup: microfluidic device with DRG culture on the somal side growing towards the axonal side. EV labelled with PKH26 lipophilic dye (red) added to the axonal side in close contact with nerve terminals. B Representative images of sensory neurons stained against calcitonin gene-related peptide (CGRP, green) with internalized EV (red) in the axonal compartment in the microfluidic devices; scale bar: 50 µm. C Representative images of EV labelled internalization by sensory neurons. Live imaging was performed under controlled temperature and CO₂ over 0, 1 and 2 h showing the sensory neurons in brightfield (grey) and internalized EV (red); scale bar: 25 µm. Superimposed tracing of total neurites (grey) and neurites with internalized EV (black 1 h, orange 2 h), attained with Simple Neurite Tracer plugin, Fiji software, are shown in the far right. D Representative images of EV labelled internalization by sensory neurons after 24 h incubation. Images from axonal side, microchannels and somal side of the microfluidic device. Brightfield images showing the sensory neurons extensions and EV in red. White asterisks indicate sensory neurons without internalized EV. Orthogonal projections depicting the selective internalization of the EV (red); scale bar: 25 µm top view and 1 µm orthogonal view. Lower row shows zoom in of the indicated squares 1, 2 and 3. E Quantification of the EV internalization throughout time. Data represented as percentage of total neurites (mean ± SD), *p ≤ 0.05