Live-imaging of axonal cargoes in human iPSC-derived neurons or mouse primary neurons
Dan Dou, C. Alexander Boecker, Erika L.F. Holzbaur
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
Steps
Live-imaging of axonal cargoes in human iPSC-derived neurons or mouse primary neurons
Image primary mouse cortical neurons on DIV7. Image human iNeurons on DIV21.
Replace culture media with low fluorescence imaging media.
For primary mouse neurons, use Hibernate E medium supplemented with
| A | B |
|---|---|
| B-27 | 2% |
| GlutaMAX | 2 mM |
For iNeurons, use Hibernate A medium supplemented with
| A | B |
|---|---|
| BDNF | 10 ng/mL |
| NT-3 | 10 ng/mL |
| B-27 | 2% |
Image using spinning disk confocal microscope under 60x magnification (oil immersion objective).
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.
Acquire time lapse recordings at a frame rate of 1 frame per second for 0h 5m 0s.
