Culture and transfection of iPSC-derived neurons for live-imaging of axonal cargoes
Dan Dou, Erika L.F. Holzbaur, Alexander Boecker
Abstract
Here, we plate, culture, and transfect human iPSC-derived excitatory glutamatergic neurons for the purpose of observing transport of axonal cargoes under spinning disk confocal microscopy. Protocol is largely as previously described (Boecker et al., 2020, 2021; Fernandopulle et al., 2018). For preceding differentiation of neurons, see “Protocol: Piggybac-mediated stable expression of NGN2 in iPSCs for differentiation into excitatory glutamatergic neurons” and “Protocol: iNeuron differentiation from human iPSCs.”
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Culture and transfection of iPSCderived neurons for live-imaging of axonal cargoes
In advance, prepare 10x PLO stock.
| A | B |
|---|---|
| PLO | 50 mg |
| 0.1M borate buffer | 50 mL |
The day before plating, coat imaging dishes with 1x PLO solution (10x PLO stock diluted in ddH2O).
The day of plating, remove PLO solution from imaging dishes and wash twice with ddH2O.
Add 2mL of iNeuron culture media.
BrainPhys supplemented with
| A | B |
|---|---|
| BDNF | 10 ng/mL |
| NT-3 | 10 ng/mL |
| Laminin | 1 μg/mL |
| B-27 supplement | 1x |
Place dishes in cell culture incubator for >0h 30m 0s.
Rapidly thaw cryopreserved iNeurons in 37°C water bath.
Centrifuge to remove freezing media and resuspend cell pellet in iNeuron culture media.
BrainPhys supplemented with
| A | B |
|---|---|
| BDNF | 10 ng/mL |
| NT-3 | 10 ng/mL |
| Laminin | 1 μg/mL |
| B-27 supplement | 1x |
Count cells and plate 300k neurons per 35 mm imaging dish.
Add cells dropwise to the center area of the dish (so that they sink onto the glass, PLO-coated center).
For Piggybac-delivered NGN2 neurons, include 10micromolar (µM) 5-Fluoro-2′-deoxyuridine and 10micromolar (µM) uridine at the time of plating to prevent survival of mitotic cells.
Store neurons in cell culture incubator. Perform partial change of iNeuron media twice per week.
On DIV18 (~72h 0m 0s prior to imaging), transfect iNeurons for imaging.
