May 18, 2025
Primary cortical neuron nucleofection Amaxa Lonza
- Sierra Palumbos1,
- Erika Holzbaur1
- 1University of Pennsylvania
Protocol Citation: Sierra Palumbos, Erika Holzbaur 2025. Primary cortical neuron nucleofection Amaxa Lonza. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3wo9zv25/v1
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: May 16, 2025
Last Modified: May 18, 2025
Protocol Integer ID: 218444
Funders Acknowledgements:
National Institute of Neurological Disorders and Stroke
Grant ID: R01-NS060698
National Institute of Neurological Disorders and Stroke
Grant ID: F32-NS129586
Michael J. Fox Foundation for Parkinson's Research (MJFF)
Grant ID: MJFF-021130
Michael J. Fox Foundation for Parkinson's Research (MJFF)
Grant ID: MJFF-15100
Michael J. Fox Foundation for Parkinson's Research (MJFF)
Grant ID: MJFF-019411
Abstract
Protocol to nucleofect primary murine cortical neurons. Protocol used in Palumbos et al., 2025
Protocol is adapted from protocol provided by LONZA- "Amaxa mouse neuron nucleofector kit."
Materials
Prepare INB nucleofection solution (ahead of time)
Composition:
- 135 mM KCl: 13.5 mL of 1M KCl
- 0.2 mM CaCl2: 20 μL of 1M CaCl2
- 2 mM MgCl2: 200 μL of 1M MgCl2
- 10 mM HEPES pH 7.3: 1 mL of 1M HEPES
- 5 mM EGTA: 1 mL of 0.5M EGTA
- 84.28 mL ddH2O
Total: 100 mL
Prepare INB nucleofection solution ahead of time
Prepare INB nucleofection solution ahead of time
Make INB buffer
| Composition | Volume | of Stock | |
| 135 mM KCl | 13.5 mL | 1M KCl | |
| 0.2 mM CaCl2 | 20 μL | 1M CaCl2 | |
| 2 mM MgCl2 | 200 μL | 1M MgCl2 | |
| 10 mM HEPES pH 7.3 | 1 mL | 1M HEPES | |
| 5 mM EGTA | 1 mL | 0.5M EGTA | |
| 84.28 mL | ddH2O | ||
| Total | 100mL |
Aliquot into 500µL
Note
Aliquots should not be refrozen
Store at -80
Isolate Primary Cortical Neurons for Nucleofection
Isolate Primary Cortical Neurons for Nucleofection
Follow previously published protocol to isolate cortical neurons from e15.5 mice.
Determine concentration of neurons.
Collect 3 million neurons in a 15 mL conical tube for each nucleofection.
Pellet cells at 1.1k RPM for 5 minutes.
Prepare Nucleofection Mix Solutions
Prepare Nucleofection Mix Solutions
Prepare 100 µL of INB per nucleofection.
Add appropriate amount of DNA.
For shRNA, add 3 µg DNA/million neurons.
For reporters, add 1 µg DNA/million neurons.
Resuspend Pelleted Neurons
Resuspend Pelleted Neurons
Remove remaining attachment media using P1000 and P200 (sequential to get total volume without perturbing pellet).
Resuspend pelleted neurons in 100 µL of INB + DNA.
Mix using P200.
Note
2X up and down should be sufficient- you do not want to be too harsh
Transfer cell suspension to nucleofection cuvette.
Nucleofect Neurons
Nucleofect Neurons
Start Amaxa Nucleofector II Device (VPG-1001) and load experiment program 0-005.
Ensure that cell suspension covers the bottom of the cuvette.
Place nucleofection cuvette with closed lid into retainer of nucleofector.
Groove containing solution should align with indents.
Nucleofect with program 0-005 by pressing “Start”.
Carefully remove cuvette.
Immediately add pre-warmed media to nucleofected neurons.
For 3 million cells, add 200 µL so total volume is 300 µL.
Plate desired amount of cells.
Note
Nucleofected neurons take longer to attach. Wait 24hours before changing from attachment to maintenance media
For 10-cm dish, add 5 million.
For imaging dish, add 500K.