Nocodazole-based microtubule dynamics analysis

Nocodazole to final concentration of 50 pM was administered in 400 μl culture media. Concentration of Nocodazole was determined after series of titrations and 50 pM was found to be the optimum to keep the MTs intact in order to perform MT dynamics analyses. Time-lapse images of growth cones were acquired for 1 min with 2 s intervals before and 5 min after Nocodazole administration, using a Yokogawa CSU-X1M 5000 spinning disk confocal on a Zeiss inverted motorized microscope with a Zeiss 63× Plan Apo 1.4 NA and a Hamamatsu ORCA R2 CCD camera. MT dynamics were assessed, as described [7 (link)].

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Erdogan B., Cammarata G.M., Lee E.J., Pratt B.C., Francl A.F., Rutherford E.L, & Lowery L.A. (2017). The microtubule plus-end-tracking protein TACC3 promotes persistent axon outgrowth and mediates responses to axon guidance signals during development. Neural Development, 12, 3.

Publication 2017

CSU-X1M 5000 ORCA R2 CCD camera

Culture media Growth cones Microscope Nocodazole Orca Titrations

Corresponding Organization :

Other organizations : Boston College

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Variable analysis

independent variables

Nocodazole concentration (final concentration of 50 pM)

dependent variables

Microtubule (MT) dynamics

control variables

Culture media volume (400 μl)
Imaging technique (Yokogawa CSU-X1M 5000 spinning disk confocal on a Zeiss inverted motorized microscope with a Zeiss 63× Plan Apo 1.4 NA and a Hamamatsu ORCA R2 CCD camera)
Imaging duration (1 min before and 5 min after Nocodazole administration)
Imaging interval (2 s)

controls

Positive control: Not mentioned
Negative control: Not mentioned

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