TITLE: Preparation of primary hippocampal neurons
AUTHORS: Arpine Sokratian, yuan.yuan, andrew.west west

[DESCRIPTION]
This protocol details preparation of the primary hippocampal neuron culture. The protocol involves extraction ofdissecting hippocampi from rodent embryos P1 from nTg or transgenic mice, enzymatic digestion to dissociate cells, and seeding onto poly-D-lysine-coated dishes. 

[STEPS]
SECTION: Plate preparation
1. Wash 48 well plates   with distilled H2O   

SECTION: Plate preparation
2. Treat each well with 0.5 mL PDL at 37 °C for 60 min.

SECTION: Plate preparation
3. Wash with distilled H2O.
SECTION: Plate preparation
4. Place 0.5 mL plating media into each well at 37 °C. 
 


SECTION: Dissection
5. Collect the hippocampi of 1-day postnatal CD1 mice pups.
SECTION: Dissection
6. Clean surgical instrument with 70% ethanol (Sharp fine scissors 14060-11 F.S.T, Sekmen Forceps, 11008-15 F.S.T).
SECTION: Dissection
7. Cut off head and using scissors, cut skin of the top of the head laterally.
SECTION: Dissection
8. Stabilizing head with tweezers, use another set of tweezers to pull skin to the side.
SECTION: Dissection
9. Cut through the bone.
SECTION: Dissection
10. Using a spoon, scoop brain from underneath and place into culture dish.
SECTION: Dissection
11. Separate the two hemispheres at the interhemispheric fissure from the brainstem.
SECTION: Dissection
12. Cut off the olfactory bulbs.
SECTION: Dissection
13. Carefully pull off the meninges but leave connected at the bottom.
SECTION: Dissection
14. Flip so that the bottom of the brain is facing up.
SECTION: Dissection
15. Completely pull off the meninges and associated tissues.
SECTION: Dissection
16. Hippocampus should be visible and make two cuts at either end.
SECTION: Dissection
17. Flip out hippocampus.
SECTION: Dissection
18. Trim hippocampus so that additional tissue is discarded.
SECTION: Dissection
19. Dissect hippocampus and using one pair of tweezers to push the tissue onto another, place hippocampus into the 15-mL tube with the 10 mL L-glutamin.

SECTION: Cell culture
20. Wash with HBSS by pipetting to aspirate and NOT vacuuming.

SECTION: Cell culture
21. Leave 1 mL HBSS in tube.

SECTION: Cell culture
22. After filtering the enzyme solution with rotator, add all 10 mL of solution to the tube.

SECTION: Cell culture
23. Incubate at 37 °C for around 45 min and no longer than 1 hour.

SECTION: Cell culture
24. Remove media and leave 1 mL.

SECTION: Cell culture
25. Pipette 10 mL plating media and 50 µL DNAse (DNAse stock is 50 µg/mL) into a 15-mL tube and invert 2-3 times.

SECTION: Cell culture
26. Pipette 10 mL DNAse solution to the 1 mL digestion solution with hippocampi and invert 2-3 times.

SECTION: Cell culture
27. Remove all media.
SECTION: Cell culture
28. Wash with 10 mL plating media.

SECTION: Cell culture
29. Wash with HBSS by pipetting to aspirate and NOT vacuuming.
SECTION: Cell culture
30. Remove HBSS and leave 1 mL with HBSS with hippocampi.

SECTION: Cell culture
31. Resuspend hippocampi with P1000 filter tip and pipet up and down 20 times until obvious chunks disappear.
SECTION: Cell culture
32. Add 5 mL plating media.

SECTION: Cell culture
33. Pass cells through strainer with 40 μm mesh size.
SECTION: Cell culture
34. Count cells and plate 50,000 cells per well for a 48 well plate, and 25,000 cells per well for a 96 well plate. 
SECTION: Cell culture
35. Leave plates in 37 °C incubator for 720 min.
SECTION: Cell culture
36. Remove the plating media and swap with culture media containing neurobasal media supplemented with B27 and 0.5 millimolar (mM) L-glutamine.
SECTION: Cell culture
37. Culture the cells for an additional 7 days before use. At day-in-vitro (DIV) 10, add fibril strains to each well of neurons to a final estimated concentration of 0.62 nanomolar (nM) (1 µg/mL).
SECTION: Plate preparation
1.1. Wash 48 well plates with 0.5 mL distilled H2O. (1/3)

SECTION: Plate preparation
1.2. Wash 48 well plates with 0.5 mL distilled H2O. (2/3)
SECTION: Plate preparation
1.3. Wash 48 well plates with 0.5 mL distilled H2O. (3/3)
SECTION: Plate preparation
3.1. Wash with 0.5 mL distilled H2O. (1/3)
SECTION: Plate preparation
3.2. Wash with 0.5 mL distilled H2O. (2/3)
SECTION: Plate preparation
3.3. Wash with 0.5 mL distilled H2O. (3/3)
SECTION: Cell culture
20.1. Wash with 10 mL HBSS by pipetting to aspirate and NOT vacuuming. (1/2)

SECTION: Cell culture
20.2. Wash with 10 mL HBSS by pipetting to aspirate and NOT vacuuming. (2/2)
SECTION: Cell culture
29.1. Wash with 10 mL HBSS by pipetting to aspirate and NOT vacuuming. (1/2)
SECTION: Cell culture
29.2. Wash with 10 mL HBSS by pipetting to aspirate and NOT vacuuming. (2/2)
SECTION: Cell culture
34.1. Using cell counter.
Place glass slip on top of slide and pipette 10 µL cell solution under slip. 
Count number of cells in one 4x4 grid (live cells appear round with dark ring and transparent inside)→# x 104 cells/mL.
SECTION: Cell culture
34.2. Make sure not to add a small volume of cells to a large volume of plating media, aim for 250 µL of cell solution per well for a 48 well plate.