TITLE: Primary culture cortical / hippocampal neurons E15-17 mouse - PFF testing
AUTHORS: Rong Chen, Sabina Marciano, Roberta Marongiu, Ted Dawson

[DESCRIPTION]
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

[STEPS]
SECTION: Dissection/Culture
12. Dissect the cortex/hippocampus and separate and remove the soft membrane and blood vessels.
SECTION: Dissection/Culture
13. Collect all the cortices/hippocampus in 30 mL  PBS  on ice. 

SECTION: Dissection/Culture
14. Transfer the cortices/hippocampus to a 15 ml tube containing 9 mL  trypsin–EDTA (0.25%) and incubate at 37 °C  for 15 min , gently shake every 5 min.

SECTION: Dissection/Culture
15. Dissociate the cortices/hippocampus by triturating with a 10 mL serological pipette 10–15 times, or until no chunks are left.
SECTION: Dissection/Culture
16. Centrifuge the dissociated cortices/hippocampus at 1500 rpm for 5 min. 

SECTION: Dissection/Culture
17. Resuspend the pellet in 10 mL DMEM high glucose with 10% FBS.

SECTION: Dissection/Culture
18. Triturate the cell suspension 10 times with a 1ml pipette.
SECTION: Dissection/Culture
19. Centrifuge at 1500 rpm for 5 min

SECTION: Dissection/Culture
20. Resuspend the pellet in 10 mL Neurobasal-A Medium with 2% B-27 Supplement (50X).

SECTION: Dissection/Culture
21. Count the cells and plate in 12 well plate 450.000/well.
SECTION: Dissection/Culture
22. Change medium after 5760 min (4 days).

SECTION: Dissection/Culture
11. Dissect and collect the brains in ice cold HBSS.
SECTION: Dissection/Culture
10. Dissect their embryos and collect in ice cold HBSS (no phenol red, no Ca/Mg).
SECTION: Dissection/Culture
9. Kill pregnant mice (E15-17) by CO2 intoxication and cervical dislocation.
SECTION: Coating coverslip
1. 
Reconstitute in 4 mL 4mL distilled sterile water.

SECTION: Coating coverslip
2. Aliquot 100 µL and store at -20 °C. 

SECTION: Coating coverslip
6. Wash x3 with sterile water.

SECTION: Coating coverslip
5. Incubate the coverslips with Poly-D-Lysine (1 mL /12well plate) for 30 min  in incubator.

SECTION: Coating coverslip
4. Sterilize autoclaved coverslips under UV for 20 min.

SECTION: Coating coverslip
3. Dilute the 100 µL  aliquot in 25 mL  PBS no Ca/mg.

SECTION: Coating coverslip
8. Add 500 µL  Neurobasal A to the well and place in incubator.

SECTION: Coating coverslip
7. Dry out under the hood.

SECTION: PFF Incubation
23. When neurons are at DIV7 proceed with PFF infection  
N.B: If PFFs are added at DIV10 the aggregation is quicker.
SECTION: PFF Incubation
24. Dilute 5mg/mL PFFs to 0.1mg/mL (20uL PFF+980uL PBS).
SECTION: PFF Incubation
25. 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.
SECTION: PFF Incubation
26. Let it settle for 1 min. 

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