Jan 09, 2026

Public workspacePostnatal primary hippocampal neuronal cultures

DOI
https://dx.doi.org/10.17504/protocols.io.kxygx4d4dl8j/v1
  • Akio Mori1,
  • Robert Edwards1
  • 1University of California San Francisco
Akio Mori
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.kxygx4d4dl8j/v1
Protocol Citation: Akio Mori, Robert Edwards 2026. Postnatal primary hippocampal neuronal cultures. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx4d4dl8j/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: November 20, 2025
Last Modified: January 12, 2026
Protocol Integer ID: 233108
Keywords: postnatal primary hippocampal neuronal cultures this protocol, preparation of postnatal primary hippocampal, postnatal primary hippocampal, neuronal culture, preparation
Abstract
This protocol describes the preparation of postnatal primary hippocampal neuronal cultures.
Materials
  • Dissection solution (HBSS++) 500 mL
AB
491 mLHank’s Balanced Salt Solution (HBSS) (UCSF Cell Culture Facility)
5 mL10 mM HEPES (UCSF Cell Culture Facility)
4 mL20 mM D-(+)-Glucose solution (G8769, Sigma)

  • Serum Neuronal Media (SNM) 25 mL

AB
22.8 mLMinimal Essential Medium (MEM) (11090081, Gibco)
213 μL45% D-(+)-Glucose solution (G8769, Sigma)
250 μLGlutamax (3505006, Gibco)
(Sterilize with a 0.22 μm syringe filter, and add the following reagents)
1.25 mLFBS (SH30070.02, Cytiva)
500 μLB27 (17504-044, Gibco)

  • Dissociation
Trypsin 0.25% (15090-046, Gibco)

  • Neurobasal Media (NBM) 25 mL
AB
24.175 mL Neurolbasal Media (21103-049, Gibco)
325 μLGlutamax (35050061, Gibco)
(Sterilize with a0.22 μm syringe filter, and add the following reagents)
500 μLB27 (17504-044, Gibco)
  • Other Supplies
Warner Instruments 1.5 Glass coverslip 25 mm round, 54-0715
Cloning cylinders, 10×10 mm (CLS-1777-03, Chemglass Life Sciences)
Poly-L-lysine for coating (P2636, Sigma-Aldrich)

Troubleshooting
Plate and coverslip preparation and coating
Sterilize coverslips and clonal rings by submerging them in 70% (v/v) ethanol and briefly flaming them over a Bunsen burner.
Place sterile coverslips into each well of a 6-well plate.
Prepare clonal rings with grease applied to the bottom, and place each ring in the center of a coverslip.
Add poly-L-lysine (PLL) solution to cover the surface of each well (with coverslip and clonal ring in place). Incubate for 3 hours at 37°C in a 5% CO₂ incubator.
Incubation
Remove PLL solution and wash the wells three times with sterile PBS.
Dissection and Dissociation
Dissect hippocampi from postnatal day 0–1 (P0-P1) mouse pups (appropriate genotypes) in HBSS++ solution.
Transfer tissue to 4.5 mL of HBSS++ containing 500 μL of 0.25% trypsin (15090-046, Gibco) in a 15 mL tube.
Incubate tissue in a 37°C water bath for 17 minutes for enzymatic digestion.
Wash tissue three times with SNM medium to remove trypsin.
Mechanical Dissociation
Add 1 mL of SNM to the tissue.
Using a glass Pasteur pipette of original diameter, triturate 3–4 times.
Flame the pipette to reduce its diameter by half. Triturate the tissue 5 times with the 1/2-diameter pipette.
Flame the pipette again to reduce its diameter to 1/4 of the original size. Triturate 5 times with the 1/4-diameter pipette.
Collect the 1 mL suspension of the resulting single cells and transfer to a 1.5 mL tube.
Cell counting and plating
Take 10 μL of the cell suspension and transfer to a 0.5 mL tube. Add Trypan Blue solution and count viable cells using a hemocytometer.
Plate dissociated neurons onto PLL-coated coverslips inside the 6-well plate at a density of 30,000–40,000 viable cells per well.
Incubate at 37°C, 5% CO₂.
On the day after plating, carefully replace 75% of the SNM with fresh, pre-warmed Neurobasal Medium (NBM) in each well using a 1 mL pipette.