Aug 04, 2024
Preparation of primary hippocampal neurons
- 1Duke Univeristy;
- 2Duke University
- West lab protocols
Protocol Citation: Arpine Sokratian, yuan.yuan, andrew.west west 2024. Preparation of primary hippocampal neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkk241l5r/v1
Manuscript citation:
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: May 29, 2022
Last Modified: August 05, 2024
Protocol Integer ID: 63449
Keywords: primary neuron, mouse hippocampus, ASAPCRN
Funders Acknowledgements:
ASAP
Grant ID: 020527
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
451-951.docx
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Materials
Prepare solutions for plating
Borate buffer:
- 50 millimolar (mM) , 3.09 g/L , 8.5
- Sterilize with 0.2 um filter.
Poly D lysine (PDL) hydrobromide (gibco, 0.1mg/ml, REF: A3890401. Life Technologies Corporation 3175 Staley Rd., Grand Island, NY 14072, USA):
| A | B | |
| 40x stock | 2 mg/mL | |
| Borate buffer pH 8.5 | 100 mg/50 mL |
- For 1x, dilute 40x with borate buffer.
- Sterilize with 0.2 um filter.
- Store in -20 °C .
Prepare solutions for dissection
Note
The same filter unit can be applied for each step of dissection
HBSS (Hanks’ Balanced Salt Solution 1X) HBSSGibco - Thermo FischerCatalog #14025-092
| A | B | |
| HBSS | 490 mL | |
| HEPES pH 7.4 | 6 mL | |
| pen/strep | 3 mL | |
| 100 mM pyruvic acid | 6 mL | |
| distilled H2O | 100 mL | |
| Total | 600 mL |
Filter (VWR Vacuum Filtration 250 ML 0.45 µm PES FILTER UNIT, Made in China Manufactured for VWR International, LLC 100 Matsonford Rd, Radnor, PA 19087)
Enzyme solution
| A | B | |
| HBSS | 10 mL | |
| Papain suspension | 200 µL | |
| L-cysteine | 2 mg | |
| 0.5 mM EDTA | 22 µL |
- Rotate for 00:30:00 .
- Filter (Millex-GP. Syringe-driven Filter Unit. 33mm, Pes Membrane, 0.22 µm, Sterilized. Merck Millipore Ltd. Tullagreen, Carrigtwohill, Co. Cork, IRELAND. Rev. 07/20).
Plating media (containing neurobasal media, supplemented with 5% FBS, 1x B27 supplement (ThermoFisher,17504044), 0.5 millimolar (mM) L-glutamine, and 100 unit per mL of Penicillin-Streptomycin)
| A | B | |
| Neurobasal | 183 mL | |
| FBS | 10 mL | |
| GlutaMAX | 2 mL | |
| B27 | 4 mL | |
| Penicillin-Streptomycin | 1 mL |
- Filter (VWR Vacuum Filtration 250ML 0.45 µm PES FILTER UNIT, Made in China Manufactured for VWR International, LLC 100 Matsonford Rd. Radnor, PA 19087) and keep in 4 °C .
- Warm Overnight /2 hours before in incubator in T75 flask (Thermo Fisher Scientific, NuncTM EasYFlaskTM 25 cm2 NunclonTM Delta Surface.Nunc A/S. Kamstrupvej 90. P.O. Box 280 DK-4000 Roskilde. Denmark) to calibrate pH.
Neuronal/culture media (containing neurobasal media supplemented with B27 and 0.5 millimolar (mM) L-glutamine)
| A | B | |
| Neurobasal | 193 mL | |
| GlutaMAX | 2 mL | |
| B27 | 4 mL |
- Filter (VWR Vacuum Filtration 250ML 0.45 µm PES FILTER UNIT, Made in China Manufactured for VWR International, LLC 100 Matsonford Rd. Radnor, PA 19087).
- Warm Overnight /2 hours before in incubator in T75 flask to calibrate pH.
Materials
Poly-D-LysineThermo Fisher ScientificCatalog #A3890401
HBSSGibco - Thermo FischerCatalog #14025-092
PapainWorthington Biochemical CorporationCatalog #LS003126
B-27 SupplementGibco - Thermo FischerCatalog #17504044
Neurobasal™ Medium, minus phenol redThermo FisherCatalog #12348017
Glutamax (100x)Gibco - Thermo FischerCatalog #35050-061
Protocol materials
Neurobasal™ Medium, minus phenol redThermo FisherCatalog #12348017
Glutamax (100x)Gibco - Thermo Fisher ScientificCatalog #35050-061
Poly-D-LysineThermo Fisher ScientificCatalog #A3890401
HBSSGibco - Thermo Fisher ScientificCatalog #14025-092
PapainWorthington Biochemical CorporationCatalog #LS003126
B-27 SupplementGibco - Thermo Fisher ScientificCatalog #17504044
HBSSGibco - Thermo Fisher ScientificCatalog #14025-092
Nunc™ Cell-Culture Treated Multidishes 48 wellThermo ScientificCatalog #12565322
Water sterile-filteredMerck MilliporeSigma (Sigma-Aldrich)Catalog #RNBK1827
Plate preparation
Plate preparation
Wash 48 well platesNunc™ Cell-Culture Treated Multidishes 48 wellGibco, ThermoFisherCatalog #12565322 with distilled H2O Water sterile-filteredGibco, ThermoFisherCatalog #RNBK1827
Wash 48 well plates with 0.5 mL distilled H2O. (1/3)
Wash 48 well plates with 0.5 mL distilled H2O. (2/3)
Wash 48 well plates with 0.5 mL distilled H2O. (3/3)
Treat each well with 0.5 mL PDL at 37 °C for 01:00:00 .
1h
Wash with distilled H2O.
Wash with 0.5 mL distilled H2O. (1/3)
Wash with 0.5 mL distilled H2O. (2/3)
Wash with 0.5 mL distilled H2O. (3/3)
Place 0.5 mL plating media into each well at 37 °C .
Note
Note: This should be done right before dissection.
Dissection
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 10 mL L-glutamin.
Cell culture
Cell culture
12h 45m
12h 45m
Wash with HBSS by pipetting to aspirate and NOT vacuuming.
Wash with 10 mL HBSS by pipetting to aspirate and NOT vacuuming. (1/2)
Wash with 10 mL HBSS by pipetting to aspirate and NOT vacuuming. (2/2)
Leave 1 mL HBSS in tube.
After filtering the enzyme solution with rotator, add all 10 mL of solution to the tube.
Incubate at 37 °C for around 00:45:00 and no longer than 1 hour.
45m
Remove media and leave 1 mL .
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.
Pipette 10 mL DNAse solution to the 1 mL digestion solution with hippocampi and invert 2-3 times.
Remove all media.
Wash with 10 mL plating media.
Wash with HBSS by pipetting to aspirate and NOT vacuuming.
Wash with 10 mL HBSS by pipetting to aspirate and NOT vacuuming. (1/2)
Wash with 10 mL HBSS by pipetting to aspirate and NOT vacuuming. (2/2)
Remove HBSS and leave 1 mL with HBSS with hippocampi.
Resuspend hippocampi with P1000 filter tip and pipet up and down 20 times until obvious chunks disappear.
Add 5 mL 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 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.
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.
Leave plates in 37 °C incubator for 12:00:00 .
12h
Remove the plating media and swap with culture media containing neurobasal media supplemented with B27 and 0.5 millimolar (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 0.62 nanomolar (nM) (1 µg/mL ).