May 23, 2023

Public workspaceLive-imaging of axonal cargoes in human iPSC-derived neurons or mouse primary neurons

Cell reports
DOI
dx.doi.org/10.17504/protocols.io.rm7vzb3r4vx1/v1
  • Dan Dou1,2,
  • C. Alexander Boecker3,
  • Erika L.F. Holzbaur1,2
  • 1Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA;
  • 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, USA;
  • 3Department of Neurology, University Medical Center Goettingen, 37077 Goettingen, Germany
Dan Dou
Open access
DOI: dx.doi.org/10.17504/protocols.io.rm7vzb3r4vx1/v1
External link: https://doi.org/10.1016/j.celrep.2023.112448
Protocol Citation: Dan Dou, C. Alexander Boecker, Erika L.F. Holzbaur 2023. Live-imaging of axonal cargoes in human iPSC-derived neurons or mouse primary neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzb3r4vx1/v1
Manuscript citation:
Boecker, C.A., Olenick, M.A., Gallagher, E.R., Ward, M.E., and Holzbaur, E.L.F. (2020). ToolBox: Live Imaging of intracellular organelle transport in induced pluripotent stem cell‐derived neurons. Traffic 21, 138–155. Kaech, S., and Banker, G. (2006). Culturing hippocampal neurons. Nat. Protoc. 1, 2406–2415.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 13, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 71302
Keywords: iPSC, iNeuron, live-imaging, axon, confocal, ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: ASAP-000350
Abstract
Here, we describe procedure and equipment used for live-imaging of axonal cargoes. This was performed both using primary mouse cortical neurons and human iPSCderived excitatory glutamatergic neurons. Equipment and software used varied based on laboratory site and scheduled upgrades to microscopy equipment during the course of this study.
Attachments
551-1147.pdf
551-1147.pdf
125KB
Guidelines
Citations:

  • Boecker, C.A., Olenick, M.A., Gallagher, E.R., Ward, M.E., and Holzbaur, E.L.F. (2020). ToolBox: Live Imaging of intracellular organelle transport in induced pluripotent stem cell‐derived neurons. Traffic 21, 138–155.
  • Kaech, S., and Banker, G. (2006). Culturing hippocampal neurons. Nat. Protoc. 1, 2406–2415.
Materials

Reagents

  • Hibernate E low fluorescence media (CATALOG)
  • ReagentGlutaMAX™ SupplementGibco - Thermo FisherCatalog #35050061
  • ReagentB-27™ Supplement (50X), serum freeGibco - Thermo FisherCatalog #17504044
  • Hibernate A low fluorescence media (BrainBits, Cat# HALF)
  • ReagentRecombinant Human NT-3peprotechCatalog #450-03
  • ReagentRecombinant Human/Murine/Rat BDNFpeprotechCatalog #450-02

Equipment

  • Heated environmental imaging chamber (Temperature37 °C )
  • Spinning disk confocal microscope (see Materials and Methods for specific systems and cameras used)
  • 60x 1.40 NA oil immersion objective
  • VisiView software





Safety warnings
Attention
Investigators should be trained and familiar with the confocal microscope to avoid eye damage from lasers.
Live-imaging of axonal cargoes in human iPSC-derived neurons or mouse primary neurons
Live-imaging of axonal cargoes in human iPSC-derived neurons or mouse primary neurons
5m
5m

Note
Please refer “Protocol: Primary neuron culture for live-imaging of axonal cargoes” and “Protocol: Culture and transfection of iPSC-derived neurons for live-imaging of axonal cargoes” for plating and transfection instructions.

Image primary mouse cortical neurons on DIV7. Image human iNeurons on DIV21.
Imaging
Replace culture media with low fluorescence imaging media.

For primary mouse neurons, use Hibernate E medium supplemented with

AB
B-272%
GlutaMAX2 mM


For iNeurons, use Hibernate A medium supplemented with

AB
BDNF10 ng/mL
NT-310 ng/mL
B-272%

Image using spinning disk confocal microscope under 60x magnification (oil immersion objective).
Note
See “Materials and Methods” for specific microscopes and cameras used.

Imaging
Identify axons of transfected neurons based on morphological parameters. (Boecker et al., 2020; Kaech and Banker, 2006). For example, axons can most reliably be identified by their length and should span over at least 500 µm.
Imaging
Acquire time lapse recordings at a frame rate of 1 frame per second for Duration00:05:00 .
Note
  • Time lapses were taken in the mid-axon, defined as >300 µm from the soma and > 100 µm from the distal axon terminal.
  • Knowledge of the pixel/micron ratio for the specific objective and camera being used is necessary for accurately measuring these distances.


5m