Live-cell imaging experiments were performed in an inverted microscope Nikon Eclipse Ti-E (Nikon), equipped with a Plan Apo VC 100x NA 1.40 oil and a Plan Apo VC 60x NA 1.40 oil objective (Nikon), a Yokogawa CSU-X1-A1 spinning disk confocal unit (Roper Scientific), a Photometrics Evolve 512 EMCCD camera (Roper Scientific) and an incubation chamber (Tokai Hit) mounted on a motorized XYZ stage (Applied Scientific Instrumentation) which were all controlled using MetaMorph (Molecular Devices) software. Coverslips were mounted in metal rings and imaged using an incubation chamber that maintains temperature and CO2 optimal for the cells (37°C and 5% CO2). Neuron live imaging was performed in full conditioned medium and fresh medium was added to COS-7 before imaging.
Time-lapse live-cell imaging of EB3-RFP was performed with a time acquisition of 1 s. NF186-RFP alone was acquired at 2 frames per second. NPY-GFP, GFP-Rab6, Rab11-GFP or Rab5-GFP, alone or in combination with NF186-RFP were acquired at 10 frames per second.
For simultaneous imaging of green and red fluorescence, we used ET-mCherry/GFP filter set (59022; Chroma) together with the DualView (DV2; Roper) equipped with the dichroic filter 565dcxr (Chroma) and HQ530/30 m emission filter (Chroma).
Time-lapse live-cell imaging of EB3-RFP was performed with a time acquisition of 1 s. NF186-RFP alone was acquired at 2 frames per second. NPY-GFP, GFP-Rab6, Rab11-GFP or Rab5-GFP, alone or in combination with NF186-RFP were acquired at 10 frames per second.
For simultaneous imaging of green and red fluorescence, we used ET-mCherry/GFP filter set (59022; Chroma) together with the DualView (DV2; Roper) equipped with the dichroic filter 565dcxr (Chroma) and HQ530/30 m emission filter (Chroma).