Axio vert a1 inverted transmitted light microscope

Neuro2a cells stably expressing doxycycline-inducible pLVX SNX32#4-resistant construct were transiently transfected with shSNX32#4 to deplete the expression of SNX32 for 48 hr (until the completion of the experiment). Following 6 hr after transfection, the cells were induced with doxycycline for expressing shSNX32 #4-resistant SNX32 and RA in reduced serum-containing media. The formation of neurites was observed using Axio Vert.A1 Inverted Transmitted Light Microscope (Carl Zeiss Microscopy GmbH) after cell fixation or live images were captured using JuLIBr inverted microscope (NanoEnTek). The JuLIBr system is equipped with a station unit that runs inside a CO₂-regulated incubator and a scope unit that runs outside the incubator. Phase-contrast images were captured every 1  min for 48 hr using a ×4 objective and a CMOS camera with a pixel length of 0.586  µm. The cells with neurites were counted using ImageJ software.

Neurite outgrowth assay was performed as previously reported (Ma et al., 2015 (link)). Briefly, Neuro2a cells were transfected with scrambled or individual siRNA SMART pools or shc002/shSNX32/shBSG clones. Following 24 hr (siRNA) or 6 hr (shRNA) of transfection, the medium was replaced with MEM containing 1% fetal bovine serum supplemented with 10 μmol/l retinoic acid (RA) for another 48 hr to induce neurite outgrowth. The formation of neurites was observed using Axio Vert.A1 Inverted Transmitted Light Microscope (Carl Zeiss Microscopy GmbH, Göttingen, Germany) after cell fixation or live images were captured using JuLIBr inverted microscope (NanoEnTek). The JuLIBr system is equipped with a station unit that runs inside a CO₂-regulated incubator and a scope unit that runs outside the incubator. Phase-contrast images were captured every 1  min for 48 hr using a ×4 objective and a CMOS camera with a pixel length of 0.586  µm. The cells with neurites were counted using ImageJ software.