May 22, 2025

Public workspacePrimary culture cortical / hippocampal neurons E15-17 mouse - PFF testing

DOI
https://dx.doi.org/10.17504/protocols.io.ewov1nemkgr2/v1
  • Rong Chen1,
  • Roberta Marongiu2,
  • Ted Dawson3,
  • Sabina Marciano4
  • 1Johns Hopkins University;
  • 2Weill Cornell Medicine;
  • 3John Hopkins University School of Medicine;
  • 4Weill Cornell medicine
Jacquelyn Haytayan
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.ewov1nemkgr2/v1
Protocol Citation: Rong Chen, Roberta Marongiu, Ted Dawson, Sabina Marciano 2025. Primary culture cortical / hippocampal neurons E15-17 mouse - PFF testing. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1nemkgr2/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: August 19, 2022
Last Modified: May 22, 2025
Protocol Integer ID: 68921
Keywords: PFF, culture, ASAPCRN, production of alpha synuclein, alpha synuclein, preparation of pre formed fibril, pre formed fibril, toxicity of pff, fibril, hippocampal neurons e15, hippocampal neuron, pff testing, pff testing this protocol, vivo experiment
Funders Acknowledgements:
Aligning Science Across Parkinsons
Abstract
This protocol is linked to the preparation of Pre Formed Fibrils by Ted Dawson's laboratory.

Tae-In Kam, Rong Chen, Ted Dawson . Production of alpha synuclein preformed fibrils (PFF). protocols.io
https://protocols.io/view/production-of-alpha-synuclein-preformed-fibrils-pf-b39rqr56

This protocol for primary culture of cortical or hippocampal neurons is meant to test the toxicity of PFF in vitro before in vivo experiment/injections.
Guidelines
Volumes calculated for n=6 brains
Materials

DMEM (15ml)
  • 1.5mL FBS heat inactivated
  • 13.5mL DMEM medium

Maintenance media (50ml)
  • 49ml Neurobasal™-A Medium
  • 1 ml B27 serum free Gibco

Reagents
ReagentFetal Bovin serum FBS (heat inactivated)Thermo Fisher ScientificCatalog #10082147
ReagentDMEM, High GlucoseLife TechnologiesCatalog #11965-092
ReagentPoly-D-LysineMP BiomedicalsCatalog #02150175-CF
ReagentNeurobasal-A MediumCatalog #10888022
ReagentB27 supplement without retinoic acid (50x)Gibco - Thermo Fisher ScientificCatalog #17504044
ReagentCoverslipElectron Microscopy SciencesCatalog #72229-01




Troubleshooting
Coating coverslip
4d 0h 30m

Reconstitute in Amount4 mL 4mL distilled sterile water.

Aliquot Amount100 µL and store at Temperature-20 °C .

Dilute the Amount100 µL aliquot in Amount25 mL PBS no Ca/mg.

Sterilize autoclaved coverslips under UV for Duration00:20:00 .

20m
Incubate the coverslips with Poly-D-Lysine (Amount1 mL /12well plate) for Duration00:30:00 in incubator.

30m
Wash x3 with sterile water.

Dry out under the hood.

Add Amount500 µL Neurobasal A to the well and place in incubator.

Dissection/Culture
4d 0h 30m
Kill pregnant mice (E15-17) by CO2 intoxication and cervical dislocation.
Dissect their embryos and collect in ice cold HBSS (no phenol red, no Ca/Mg).
Dissect and collect the brains in ice cold HBSS.
Dissect the cortex/hippocampus and separate and remove the soft membrane and blood vessels.
Collect all the cortices/hippocampus in Amount30 mL PBS TemperatureOn ice .

Transfer the cortices/hippocampus to a 15 ml tube containing Amount9 mL trypsin–EDTA (0.25%) and incubate at Temperature37 °C for Duration00:15:00 , gently shake every Duration00:05:00 .

20m
Dissociate the cortices/hippocampus by triturating with a 10 mL serological pipette 10–15 times, or until no chunks are left.
Centrifuge the dissociated cortices/hippocampus at 1500 rpm for Duration00:05:00 .

5m
Resuspend the pellet in Amount10 mL DMEM high glucose with 10% FBS.

Triturate the cell suspension 10 times with a 1ml pipette.
Centrifuge at 1500 rpm for Duration00:05:00

5m
Resuspend the pellet in Amount10 mL Neurobasal-A Medium with 2% B-27 Supplement (50X).

Count the cells and plate in 12 well plate 450.000/well.
Change medium after Duration96:00:00 (4 days).

4d
PFF Incubation
1m
When neurons are at DIV7 proceed with PFF infection
N.B: If PFFs are added at DIV10 the aggregation is quicker.
Dilute 5mg/mL PFFs to 0.1mg/mL (20uL PFF+980uL PBS).
Sonicate at amplitude 20% for a total of 60 pulses (0.5 seconds on/off cycle).
Pause briefly between every 10-12 pulses to prevent solution from heating up excessively and to avoid frothing.
Let it settle for Duration00:01:00 .

1m
Dilute to 1ug/mL (50uL PFFs+5mL neurobasal NB).
Filter in a 0.2um filter.
Replace completely the medium in well with NB+PFFs.
Incubate with PFFs for 10 days replacing half of the medium every 3 days.