Preparation of primary hippocampal neurons
Arpine Sokratian, andrew.west west, yuan.yuan
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Abstract
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.
Attachments
Steps
Plate preparation
Wash 48 well plates
Wash 48 well plates with 0.5mL distilled H2O. (1/3)
Wash 48 well plates with 0.5mL distilled H2O. (2/3)
Wash 48 well plates with 0.5mL distilled H2O. (3/3)
Treat each well with 0.5mL PDL at 37°C for 1h 0m 0s.
Wash with distilled H2O.
Wash with 0.5mL distilled H2O. (1/3)
Wash with 0.5mL distilled H2O. (2/3)
Wash with 0.5mL distilled H2O. (3/3)
Place 0.5mL plating media into each well at 37°C.
Dissection
Collect the hippocampi of 1-day postnatal CD1 mice pups.
Clean surgical instrument with 70% ethanol (Sharp fine scissors 14060-11 F.S.T, Sekmen Forceps, 11008-15 F.S.T).
Cut off head and using scissors, cut skin of the top of the head laterally.
Stabilizing head with tweezers, use another set of tweezers to pull skin to the side.
Cut through the bone.
Using a spoon, scoop brain from underneath and place into culture dish.
Separate the two hemispheres at the interhemispheric fissure from the brainstem.
Cut off the olfactory bulbs.
Carefully pull off the meninges but leave connected at the bottom.
Flip so that the bottom of the brain is facing up.
Completely pull off the meninges and associated tissues.
Hippocampus should be visible and make two cuts at either end.
Flip out hippocampus.
Trim hippocampus so that additional tissue is discarded.
Dissect hippocampus and using one pair of tweezers to push the tissue onto another, place hippocampus into the 15-mL tube with the 10mL L-glutamin.
Cell culture
Wash with HBSS by pipetting to aspirate and NOT vacuuming.
Wash with 10mL HBSS by pipetting to aspirate and NOT vacuuming. (1/2)
Wash with 10mL HBSS by pipetting to aspirate and NOT vacuuming. (2/2)
Leave 1mL HBSS in tube.
After filtering the enzyme solution with rotator, add all 10mL of solution to the tube.
Incubate at 37°C for around 0h 45m 0s and no longer than 1 hour.
Remove media and leave 1mL.
Pipette 10mL plating media and 50µL DNAse (DNAse stock is 50µg/mL) into a 15-mL tube and invert 2-3 times.
Pipette 10mL DNAse solution to the 1mL digestion solution with hippocampi and invert 2-3 times.
Remove all media.
Wash with 10mL plating media.
Wash with HBSS by pipetting to aspirate and NOT vacuuming.
Wash with 10mL HBSS by pipetting to aspirate and NOT vacuuming. (1/2)
Wash with 10mL HBSS by pipetting to aspirate and NOT vacuuming. (2/2)
Remove HBSS and leave 1mL with HBSS with hippocampi.
Resuspend hippocampi with P1000 filter tip and pipet up and down 20 times until obvious chunks disappear.
Add 5mL plating media.
Pass cells through strainer with 40 μm mesh size.
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.
Using cell counter.
- Place glass slip on top of slide and pipette
10µLcell 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.
Make sure not to add a small volume of cells to a large volume of plating media, aim for 250µL of cell solution per well for a 48 well plate.
Leave plates in 37°C incubator for 12h 0m 0s.
Remove the plating media and swap with culture media containing neurobasal media supplemented with B27 and 0.5millimolar (mM) L-glutamine.
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 0.62nanomolar (nM) (1µg/mL).
