May 23, 2023
Culture and transfection of iPSC-derived neurons for live-imaging of axonal cargoes
- Dan Dou1,2,
- 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
External link: https://doi.org/10.1016/j.celrep.2023.112448
Protocol Citation: Dan Dou, Alexander Boecker, Erika L.F. Holzbaur 2023. Culture and transfection of iPSC-derived neurons for live-imaging of axonal cargoes. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9dj4zg3e/v1
Manuscript citation:
Boecker, C.A., and Holzbaur, E.L.F. (2021). Hyperactive LRRK2 kinase impairs the trafficking of axonal autophagosomes. Autophagy 00, 1–3. 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. Fernandopulle, M.S., Prestil, R., Grunseich, C., Wang, C., Gan, L., and Ward, M.E. (2018). Transcription Factor-Mediated Differentiation of Human iPSCs into Neurons. Curr. Protoc. Cell Biol. 79, e51.
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: 71294
Keywords: iPSC, iNeuron, live-imaging, axon, ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: ASAP-000350
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.”
Attachments
550-1146.pdf
168KB
Materials
Materials
Equipment
35 mm Dish | No. 1.5 Coverslip | 20 mm Glass Diameter | Uncoated
NAME
Coverslip
TYPE
Mattek
BRAND
p35g-1-5-20-c
SKU
LINK
Reagents
- PLO (CATALOG)
- Borate buffer (CATALOG)
- BrainPhys media (CATALOG)
- NT-3 (CATALOG)
- BDNF (CATALOG)
- B-27 supplement (CATALOG)
- Mouse laminin (CATALOG)
- 5-Fluoro-2′-deoxyuridine
- Uridine
Culture and transfection of iPSCderived neurons for live-imaging of axonal cargoes
Culture and transfection of iPSCderived neurons for live-imaging of axonal cargoes
30m
30m
In advance, prepare 10x PLO stock.
| A | B | |
| PLO | 50 mg | |
| 0.1M borate buffer | 50 mL |
Note
Store 10x PLO stock at -80 °C .
The day before plating, coat imaging dishes with 1x PLO solution (10x PLO stock diluted in ddH2O).
Note
It is only necessary to fully coat the glass center of the imaging dish.
The day of plating, remove PLO solution from imaging dishes and wash twice with ddH2O.
Add 2 mL 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 >00:30:00 .
30m
Rapidly thaw cryopreserved iNeurons in 37 °C water bath.
Note
Retrieve vial to tissue culture hood when only a small amount of ice remains visible.
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 10 micromolar (µM) 5-Fluoro-2′-deoxyuridine and 10 micromolar (µM) uridine at the time of plating to prevent survival of mitotic cells.
Note
These drugs were removed 24 hours after plating.
Store neurons in cell culture incubator. Perform partial change of iNeuron media twice per week.
On DIV18 (~72:00:00 prior to imaging), transfect iNeurons for imaging.
Note
Transfection conditions may require optimization, but a typical transfection will use 4 µL Lipofectamine Stem and 1 µg of plasmid DNA. Plasmids with the PGK or EF1α promoters express best in iNeurons.
3d