Jan 14, 2026

Public workspaceCulturing Primary Hippocampal Neurons from Embryonic Rat Brain

DOI
https://dx.doi.org/10.17504/protocols.io.eq2lywrmwvx9/v1
  • Pranav Shedge1,
  • Dhrubajyoti Chowdhury2,
  • Thomas Biederer1
  • 1Yale University;
  • 2Gandhi Institute of Technology and Management (Visakhapatnam)
Maria Matos
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.eq2lywrmwvx9/v1
Protocol Citation: Pranav Shedge, Dhrubajyoti Chowdhury, Thomas Biederer 2026. Culturing Primary Hippocampal Neurons from Embryonic Rat Brain. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lywrmwvx9/v1
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: September 09, 2024
Last Modified: January 14, 2026
Protocol Integer ID: 108271
Keywords: Hippocampal Neurons, Hemocytometer, Poly-DL-Ornithine, Tissue Disaggregation, rat primary culture, primary hippocampal neurons from embryonic rat brain, culturing primary hippocampal neuron, embryonic rat hippocampus, culturing of primary neuron, embryonic rat brain, primary hippocampal, primary neuron, rat brain, neuron, culturing
Funders Acknowledgements:
ASAP
Grant ID: ASAP-020616
NIH
Grant ID: R01 DA018928 (to T.B.)
NIH
Grant ID: R01 MH119826 (to T.B.)
Disclaimer
All procedures need to be approved by the local Institutional Animal Care & Use Committee.
Abstract
This protocol details the culturing of primary neurons from the embryonic rat hippocampus.
Guidelines
All procedures on live animals should be performed in accordance with your Institutional Animal Care and Use Committee.
Materials
Dissection Media

Hanks' Buffer:

  • Dissolve ReagentHanks′ Balanced SaltsMerck MilliporeSigma (Sigma-Aldrich)Catalog #H2387 in 1L ReagentWaterMerck MilliporeSigma (Sigma-Aldrich)Catalog #W4502
  • Add 0.35g Sodium bicarbonate to obtain pH ~7.2-7.3.
  • Filter sterilize with 0.22 m filter (2x 500 ml bottles)

Hanks' Plus Buffer (500 ml):

  • 500 ml Hanks' Buffer
  • 5 mL of 1 M ReagentHEPES BufferThermo Fisher ScientificCatalog #15630-080
  • 250 l gentamycin (Final concentration 5g/ml)

Note
Store at Temperature4 °C for at least 6 months.


FUdR- 5-Fluoro-2’-deoxyuridine/uridine stock solution (10 mM in H2O):

  • 24.62 mg Reagent5-Fluoro-2′-deoxyuridineMerck MilliporeSigma (Sigma-Aldrich)Catalog #F0503
  • 24.42 mg ReagentUridineMerck MilliporeSigma (Sigma-Aldrich)Catalog #U3003
  • 10 ml milli Q water
  • Sterile filter 0.2 m, aliquot and freeze stocks at Temperature-20 °C .

Neuronal Media (50ml) – Prepare the day before or day of culture:

  • 46 ml ReagentNeurobasal mediumGibco - Thermo Fisher ScientificCatalog #21103049
  • 1 mL ReagentB-27 SupplementGibco - Thermo Fisher ScientificCatalog #17504044
  • 0.5 mL ReagentGlutamax (100x)Gibco - Thermo Fisher ScientificCatalog #35050-061
  • 2.5 ml Fetal Bovine Serum (heat-inactivated)
  • 5 l Gentamycin (10 mg/ml, Gibco 1570060)
  • Filter sterilize with 0.22 m filter
  • Store at Temperature4 °C

Note
All ingredients are thawed overnight at Temperature4 °C or TemperatureRoom temperature
Once thawed, keep at Temperature4 °C until ready to prepare the media.
Do not leave at TemperatureRoom temperature for a prolonged period of time!


Papain Solution – Prepare on day of culture:

  • 10 mL Hanks' Plus Buffer pre-warmed in Temperature37 °C water bath.
  • 200 units Papain suspension (LS003127 Worthington)

Note
  • Add papain to buffer, mix by inversion and incubate in 37 C incubator for 20-30 min.
  • Once papain is dissolved, mix by inversion and filter sterilize with 0.22 m filter.
  • Equilibrate Temperature37 °C CO2 incubator for ~30 minutes prior to use.


Feeding Media (5mL) make at 13 or 14 DIV:

  • 4.85 mL ReagentNeurobasal medium Gibco - Thermo Fisher ScientificCatalog #21103049
  • 0.1 mL ReagentB-27 SupplementGibco - Thermo Fisher ScientificCatalog #17504044
  • 50 L ReagentGlutamaxGibco - Thermo Fisher ScientificCatalog #35050-061
  • 0.5 L ReagentGentamycinGibco - Thermo Fisher ScientificCatalog #1570060
  • 5 L 10mM FUdR
  • Sterile filter 0.2 m

Poly-DL-Ornithine Stock Solution:

  • Dissolve ReagentPoly-DL-ornithine hydrobromideMerck MilliporeSigma (Sigma-Aldrich)Catalog #P0421 in sterile PBS at 1 mg/ml and store 1 ml aliquots at Temperature-80 °C .

Laminin Stock Solution:

  • Thaw ReagentLaminin, MouseCorningCatalog #354232 at Temperature4 °C overnight and then reconstitute with Hanks' buffer to a final stock concentration of 0.5 mg/ml.
  • Make 60-70 l aliquots and store at Temperature-80 °C .

Protocol materials
ReagentEthyl alcohol, PureMerck MilliporeSigma (Sigma-Aldrich)Catalog #E7023
ReagentGibco™ Neurobasal™ MediumThermo Fisher ScientificCatalog #21103049
Reagent5-Fluoro-2′-deoxyuridineMerck MilliporeSigma (Sigma-Aldrich)Catalog #F0503
ReagentNeurobasal medium Gibco - Thermo Fisher ScientificCatalog #21103049
ReagentB-27 SupplementGibco - Thermo Fisher ScientificCatalog #17504044
ReagentNeurobasal mediumGibco - Thermo Fisher ScientificCatalog #21103049
ReagentHanks′ Balanced SaltsMerck MilliporeSigma (Sigma-Aldrich)Catalog #H2387
ReagentGlutamaxGibco - Thermo Fisher ScientificCatalog #35050-061
ReagentHEPES BufferThermo Fisher ScientificCatalog #15630-080
ReagentGlutamax (100x)Gibco - Thermo Fisher ScientificCatalog #35050-061
ReagentWaterMerck MilliporeSigma (Sigma-Aldrich)Catalog #W4502
ReagentGentamycinGibco - Thermo Fisher ScientificCatalog #1570060
ReagentPoly-DL-ornithine hydrobromideMerck MilliporeSigma (Sigma-Aldrich)Catalog #P0421
ReagentLaminin, MouseCorningCatalog #354232
ReagentUridineMerck MilliporeSigma (Sigma-Aldrich)Catalog #U3003
Troubleshooting
Coverslip and Plate Preparation
3d
Begin cleaning coverslips 3-4 days prior to culture.
We use 18 mm Cover Glasses (Carolina Biological Supply, Catalog# 633013)

Critical
Place coverslips in glass container (beaker or Petri dish) containing 100% ethanol immobilized on a platform shaker and agitate for 3-4 days.
ReagentEthyl alcohol, PureMerck MilliporeSigma (Sigma-Aldrich)Catalog #E7023

Note
Agitating too vigorously can cause coverslip breakage.

3d
Rinse coverslips with ethanol 2-3 times to remove any glass dust.
Store in 100% ethanol until needed.

Note
The coverslip cleaning procedure is crucial.

Transfer coverslips to the biosafety cabinet (BSC).
Remove coverslips from ethanol and let them dry by placing them at an angle on a parafilm coated dish.
Once dried, place coverslips flat on the parafilm-coated Petri dish

Coat Coverslips and/or Plates.
Thaw Poly-DL-ornithine hydrobromide (PO) at TemperatureRoom temperature , then warm for at least Duration00:30:00 in Temperature37 °C incubator.
Note
Do not refreeze Poly-DL-ornithine hydrobromide.

30m
Thaw laminin aliquot at Temperature4 °C , always keep TemperatureOn ice .
Prepare coating solution of Amount40 μg/ml PO + Amount5 μg/ml Laminin in sterile PBS.

Note
3 mL of coating solution yields enough for approximately thirty 12 mm coverslips.


Add Amount0.1 mL coating solution to each coverslip in the parafilm coated Petri dish.

Pipetting
Incubate DurationOvernight in Temperature37 °C incubator (5% CO2).

8h
Incubation
Overnight
Temperature
Rinse 3-4 times with sterile molecular grade water (e.g., Sigma W4502)

Remove all water from coverslips or plates.
Place one coverslip per well of 24-well plate.

Add 1 ml neuronal media to each well of 24-well plate and return plate to incubator.

Note
Coating will last at least 8 days at Temperature4 °C .
If stored for more than a day before plating, add Amount1 mL ReagentGibco™ Neurobasal™ MediumThermo Fisher ScientificCatalog #21103049 per well and replace with fresh neuronal media the day of dissection prior to cell plating.

Pipetting
Dissection of Hippocampus
5m
Anesthetize pregnant rat at 18 days gestation with isoflurane in an anesthesia chamber for Duration00:05:00 or until it does not respond to toe pinch. Animal is sacrificed according to IACUC guidelines.

5m
Clean the region of the rat peritoneum with 70% ethanol.

Hold the peritoneum region with forceps and make a small cut with a scalpel. Using scissors, cut open the peritoneum region. Remove the oviduct (10 embryos approx.) without touching the intestines. Put the oviduct in petri dish containing cold Hank’s buffer on ice.

Take the petri dish with embryos to laminar flow hood. Open each placenta to remove individual embryos. Decapitate with sharp scissors and place heads in a new petri dish TemperatureOn ice containing cold Hank’s Plus buffer.
Dissect brains and transfer to a new petri dish with cold Hank’s Plus buffer TemperatureOn ice .

Under a microscope with appropriate cold light source, place the petridish with brain ventral side up. Remove meninges with fine angled (#5/15) forceps.

Remove the olfactory bulbs.
Flip the brain to dorsal side up. Separate cortical hemispheres through the midline and gently lift the cortex to access the hippocampus.
The hippocampi are at the inner edge of each hemisphere. Separate the hippocampi from the cortex using angled (#5/15) forceps.


Transfer the hippocampi to new cold Hank’s Plus buffer in a 15 mL falcon tube TemperatureOn ice . Repeat until this process has been repeated with all brains.

Tissue Disaggregation (move to biosafety cabinet)
35m
Aspirate the Hanks Plus buffer without disturbing the hippocampi. Add Amount10 mL papain solution, warmed at Temperature37 °C , to the hippocampi and incubate in 37°C incubator with loosened cap for Duration00:30:00 .

30m
Incubation
Pipetting
Temperature
After incubation, remove as much papain as possible using serological pipette. Wash three times with approximately Amount3 mL warm pre-equilibrated neuronal media.

Wash
Remove the final wash and add Amount5 mL neuronal media to the hippocampi.

Mechanically break up soft tissue by trituration 5-7 times using 10 ml pipette, avoid creating bubbles.

Do not over titrate (turbid media indicates many cells dissociated). Any non-dissociated tissue will be removed in a later step.

Centrifuge dissociated cells at Centrifigation250 rcf, 00:05:00 .

5m
Centrifigation
Aspirate media and resuspend pellet in Amount1 mL neuronal media. Add additional media to desired volume.

Pass the cell suspension through 70 um cell strainer and rinse with neuronal media.
Plating of Hippocampal Neurons
Count live cells using a hemocytometer. Averaging counts from at least two quadrants. Dilute Amount10 µL of cell suspension with Amount10 µL of trypan blue (dilution factor = 2).

  • Count live cells using a hemocytometer. Average number of cells in two quadrants x 2 (dilution factor) x 104 cells/ml.
Dilute cells to desired concentration in neuronal media and plate the appropriate volume onto by the amount you want on previously coated with Poly-DL-Ornithine / laminin coated coverslips in 24 well plate. Plate 30,000 cells/well in 24 well plates.

Cell culture maintenance
Monitor glia abundance 2-3 days after culturing.

  • If glia population is too high, treat culture with FUdR.
  • Add Amount1 µL FUdR (final concentration-Amount10 μg/ml ) for Amount1 mL neuronal media.
  • Timing of FUdR addition is determined by inspection under microscope for each culture, typically 2-3 DIV for a culture plated at 30,000 cells/well in a 24-well plate. Delay treatment if the culture does not have excessive glia.

Pipetting
At 14 DIV, replace 1/3 of neuronal medium with feeding medium; thereafter, feed cells every 2-3 days by replacing 30% media. Cells can be routinely grown until 28 DIV with minimal to no cell death.

Protocol references
D. Chowdhury and J.W. Hell (2019) ‘Ca2+/calmodulin binding to PSD-95 downregulates its palmitoylation and AMPARs in long-term depression’, Frontiers in Synaptic Neuroscience 11(6).