Metamorph

Confocal images were acquired using Carl Zeiss LSM780 laser scanning system using Argon 488 laser for puromycin. Helium-neon 594 laser was used to visualize MAP2. Oil immersion objective 63×/1.40 was used and z-stack images were captured. All confocal images for puromycin and MAP2-labeled neuronal soma and neurites were acquired under identical conditions and analyzed after blinding. Quantification of puromycin levels measured as puromycin intensity was performed using MetaMorph software (MetaMorph, version 7.8.0.0, 2013, Molecular Devices, LLC) (MetaMorph.html">http://www.moleculardevices.com/Products/Software/Meta-Imaging-Series/MetaMorph.html). Confocal z-stack images were loaded onto MetaMorph software and maximum intensity projection was generated. After background subtraction and thresholding, mask was generated around the soma and dendrite of interest (including the spines), and puromycin intensity was measured.

For migration assay, cells were seeded in 24-well Thin-Cert^TM chambers with 8 μm-pore size (Greiner bio-one (662–638)). After three days, the cells were fixed and stained by Hoechst 33342. The migrated cells were imaged and counted in 10 microscopic fields. The image were quantified and averaged by using MetaMorph (Molecular Devices).
For sphere formation assays, cells were grown in suspension culture using serum-free RPMI supplemented with B27 (Invitrogen), 20 ng/ml EGF (ProSpec CYT-218), and 10ng/ml FGF-b (ProSpec CYT-218). Sphere diameter was measured using MetaMorph (Molecular Devices).

Fluorescence microscopy was performed as previously described (Kilaru et al., 2015b ). In brief, the cells were inoculated in YG media and grown at 24 °C with 100 rpm for 24 h and placed onto a 2% agar cushion for direct observation using a motorized inverted microscope (IX81; Olympus, Hamburg, Germany), equipped with a PlanApo 100×/1.45 Oil TIRF (Olympus, Hamburg, Germany). Fluorescent tags and dyes were exited using a VS-LMS4 Laser Merge System with solid-state lasers (488 nm/50 mW or 75 mW and 561 nm/50 mW or 75 mW; Visitron Systems, Puchheim, Germany) and single images or z-Stacks, using an objective piezo (Piezosystem Jena GmbH, Jena, Germany), over 6 μm depth with a z resolution of 0.2 μm were captured with 150 ms exposure. In addition a DIC image was taken for each cell using a CoolSNAP HQ2 camera (Photometrics/Roper Scientific, Tucson, USA). Overlays of the fluorescent and DIC images as well as the pseudo-colored images were generated using MetaMorph (Molecular Devices, Wokingham, UK). All parts of the system were under the control of the software package MetaMorph (Molecular Devices, Wokingham, UK).

The cells were fixed in 1% formalin for 8 min at RT followed by treatment with 0.1% Triton X-100 in PBS. After blocking for 10 min in blocking buffer (PBS containing 1% BSA), the cells were incubated with primary antibodies in blocking buffer for 1 h at RT or overnight at 4°C. The cells were then washed three times with PBS, followed by incubation with fluorochrome-conjugated secondary antibodies for 1 h at RT. The cells were then mounted in fluorescence mounting medium (Dako). The immunofluorescently stained samples were imaged with a Olympus spinning disk super-resolution microscope (SD-OSR; Olympus) equipped with a silicon oil-immersion objective lens (UPlanSApo 60× Sil, NA 1.3; Olympus) with a 1.6× conversion lens, a sCMOS camera (ORCA-Flash 4.0 v2; Hamamatsu Photonics), appropriate filter sets for DAPI/FITC/TRITC and a motorized scanning deck. Image acquisition was set to a z-series of multiple planes with a 0.25-μm distance. Immunofluorescently stained samples were imaged using a silicon oil-immersion objective lens (UPlanSApo 60×Sil, NA 1.3; Olympus). All of the hardware was controlled with MetaMorph (Molecular Devices). The resulting 3D images were converted into 2D images by maximum intensity projection in the z-direction before the image analysis. The images were analyzed with MetaMorph (Molecular Devices).

Epifluorescence images of the mCherry-IFT27 rescue experiment were acquired with a Leica DM6000 microscope (Objective: 63×/1.4 NA Plan-Apo) equipped with a Cool SNAP HQ2 camera and controlled by MetaMorph (Molecular Devices). Confocal images were acquired with a Zeiss LSM780 microscope (Objective: 63x/1.4 NA DIC Plan-Apo) controlled by ZEN software (Zeiss). Images of 3D cultures and time-lapse were performed using a spinning disk confocal microscope, a Nikon Ti Eclipse coupled to a Yokogawa spinning disk head and an EMCCD iXon Ultra camera (Objectives 60×/1.4 NA and 100×/1.45 NA), controlled by the Andor iQ3 software (Andor). Overnight time-lapse was started 30 h after siRNA transfection or 2 h after treatment with paprotrain 5 μM (Calbiochem) and images were acquired every 1, 2 or 5 min. Image processing and analysis (cropping, rotating, brightness, contrast adjustment, color combining and measurements) were performed with ImageJ or Imaris (Bitplane). Linescans were obtained using ImageJ plot profile tool. MKLP1, MgcRacGAP and Phospho S708 MKLP1 fluorescence intensities were measured using MetaMorph (Molecular Devices).

For migration assay, 600 μl of EGM2 medium supplemented with 10% FBS were added to the lower chamber, while 5x10⁴ ECFCs in 100 μl of EGM2 medium were seeded on the upper chamber with 8 μm pore size of Costar Transwell Polycarbonate Permeable Supports (Corning, Corning, New York, USA). Six hours after incubation at 37°C with 5% CO₂, suspension cells were removed and the membranes were fixed with 4% paraformaldehyde for 15 minutes at room temperature. The migrated cells were then stained with Hoechst 33342 reagents (Sigma) for 30 minutes and counted under fluorescent microscope in five representative fields. The images were quantified using MetaMorph (Molecular Devices, Sunnyvale, CA, USA).
For in vitro tube formation assay, 96-well plate were coated with Basement Membrane Extract (BME, 3433-005-01, Trevigen Inc., Gaithersburg, MD, USA) (50 μl per well) at 37°C for an hour as described previously [33 (link)]. 1x10⁴ ECFCs in 100μl medium were placed onto the matrix and incubated at 37°C for 3 hours. The tube structures were visualized under an inverted light microscope (100 X). Five microscopic fields were captured in each group and total tube length was calculated using MetaMorph (Molecular Devices). All data were obtained from three independent experiments with triplication.