TITLE: PRIMARY NEURON CULTURE PROTOCOL
AUTHORS: Michael Lee

[DESCRIPTION]
This protocol details primary hippocampal neuron culture.

[BEFORE_START]
Before dissection

a. Dissection tools (scissors, forceps, spatulas, razor blades) should all be cleaned and autoclaved prior to use.
b. Prepare dishes or plates.
    i. Minimum of 60 min in 37 °C incubator.
c. Have solutions warmed, equilibrating, and prepared prior to starting dissection (plating media, digestion solution, digestion inhibition solution).
    i. Sterile filter digestion and inhibition solutions prior to use.
d. Flame polish autoclaved 9” Pasteur pipettes.

[STEPS]
SECTION: Primary Hippocampal Neuron Culture Protocol: Dissection
1. In laminar flow hood: have aluminum foil for mice, Kimwipes or paper towels for dissection, tools, ice bucket and Brain Bits Hibernate A (BB HA).
SECTION: Primary Hippocampal Neuron Culture Protocol: Dissection
2. Begin dissection.  
 

SECTION: Primary Hippocampal Neuron Culture Protocol: Dissection
2.1. Remove tools from alcohol.
SECTION: Primary Hippocampal Neuron Culture Protocol: Dissection
2.2. Decapitate pup/s with scissors.
SECTION: Primary Hippocampal Neuron Culture Protocol: Dissection
2.3. Use razor to make a mid-sagittal incision only penetrating the skin.
SECTION: Primary Hippocampal Neuron Culture Protocol: Dissection
2.4. Use razor to make a small mid-sagittal incision in the skull, then press down hard hemisecting the brain and skull. Push apart.
SECTION: Primary Hippocampal Neuron Culture Protocol: Dissection
2.5. Dip blunt dissecting spatulas into the wash solution. Scoop out brain hemisphere, severing the olfactory bulb for ease.
SECTION: Primary Hippocampal Neuron Culture Protocol: Dissection
2.6. Separate cortex from colliculi exposing the hippocampus.
SECTION: Primary Hippocampal Neuron Culture Protocol: Dissection
2.7. Identify the hippocampus by hallmark crescent shape, and medial, longitudinal blood vessel.
SECTION: Primary Hippocampal Neuron Culture Protocol: Dissection
2.8. Press down ventral of the hippocampus (in the crescent) with one spatula. Gently push onto this spatula with the other. The structure should cleanly slide free.
SECTION: Primary Hippocampal Neuron Culture Protocol: Dissection
2.9. Place in chilled BB HA solution.
SECTION: Primary Hippocampal Neuron Culture Protocol: Dissection
3. Keep on ice until ready to place hippocampi into warmed and sterile filtered digestion solution.
SECTION: Digestion
4. Using 10 mL serological pipette, transfer hippocampi from BB HA to digestion solution.
SECTION: Digestion
5. Incubate in 37 °C water bath for 10 min to 15 min, with intermediate mixing.

SECTION: Digestion
6. During this time:
SECTION: Digestion
6.1. Ensure plates/dishes are ready.
SECTION: Digestion
6.2. Prepare trypan blue Eppendorf tube (150 µL TB + 50 µL cells) to count.

SECTION: Inhibition + Triturate
7. Following digestion incubation, gently remove hippocampi with 10 mL serological pipette and place into 15 mL conical tube.
SECTION: Inhibition + Triturate
8. Wash hippocampi. 
SECTION: Inhibition + Triturate
9. Then add final 4 mL to 5 mL inhibition solution and triturate hippocampi gently using fire polished pasteur pipette.

SECTION: Inhibition + Triturate
10. Once triturated, allow any undissociated tissue to sink to the bottom, gently transfer remaining suspension to fresh 15 mL tube.
SECTION: Inhibition + Triturate
11. Pull 50 µL aliquot for counting, then centrifuge at 300 x g, 4 min, 4 °C.

SECTION: Inhibition + Triturate
8.1. Wash hippocampi with inhibition solution 3 mL to 4 mL. (1/3)

SECTION: Inhibition + Triturate
8.2. Wash hippocampi with inhibition solution 3 mL to 4 mL. (2/3)
SECTION: Inhibition + Triturate
8.3. Wash hippocampi with inhibition solution 3 mL to 4 mL. (3/3)
SECTION: Count cells
12. Make up the 200 µL (1:4 dilution of cells) trypan blue mixture, load 10 µL to hemacytometer, and count 4 quadrants.
SECTION: Count cells
13. Calculate desired concentration of cells/mL.
SECTION: Plate cells
14. Dilute cells with appropriate amount of pre-equilibrated plating media to get desired cell concentration and then add to dishes/plates.
SECTION: Plate cells
15. Place these in incubator and incubate for 180 min to 240 min, or 240 min before switching to growth medium (Brain Bits NbActiv4).