Wide-field images were acquired on a Nikon Eclipse Ni-E upright microscope equipped with a CFI Plan Apochromat Lambda 20× 0.75 NA objective lens (Nikon), Nikon motorized XY stage, and Andor Zyla 4.2 Plus scientific CMOS camera, driven by NIS-Elements. Alexa 647–labeled bovine tubulin, EB1-GFP (110 nM final), Tau-mCherry (20 nM final; Mooney et al., 2017 (link)), and/or Alexa 568–labeled 10 kDa dextran were supplemented to the reactions. Dark current subtraction and flat field correction were applied to both channels. A custom script written in MATLAB was used to quantify the fluorescent intensity as a function of radial distance from the MTOC (Pelletier et al., 2020 ). Alexa 568–labeled dextran showed a flat intensity profile with respect to the aster as expected for a molecule that shows no spatial variation (Supplemental Figure 4). Spot detection of EB1 comets was performed with TrackMate as described above.
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Quantitative Analysis of Microtubule Dynamics
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Corresponding Organization : Max Planck Institute of Molecular Cell Biology and Genetics
Other organizations : Institute of Science and Technology Austria, Center for Systems Biology, Harvard University, Marine Biological Laboratory, Massachusetts Institute of Technology
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- Wide-field images were acquired on a Nikon Eclipse Ni-E upright microscope equipped with a CFI Plan Apochromat Lambda 20× 0.75 NA objective lens (Nikon), Nikon motorized XY stage, and Andor Zyla 4.2 Plus scientific CMOS camera, driven by NIS-Elements.
- Alexa 647–labeled bovine tubulin, EB1-GFP (110 nM final), Tau-mCherry (20 nM final; Mooney et al., 2017 (link)), and/or Alexa 568–labeled 10 kDa dextran were supplemented to the reactions.
- Fluorescent intensity as a function of radial distance from the MTOC (Pelletier et al., 2020 (no link found)).
- Spot detection of EB1 comets was performed with TrackMate as described above.
- Dark current subtraction and flat field correction were applied to both channels.
- Alexa 568–labeled dextran showed a flat intensity profile with respect to the aster as expected for a molecule that shows no spatial variation (Supplemental Figure 4).
- Alexa 568–labeled dextran (negative control)
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