Dissociating and Culturing Neurons from Hippocampal Tissue Samples

Abstract

Source: Servello, D. et al., A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons. J. Vis. Exp. (2018).

This video demonstrates the dissociation and culture of hippocampal neurons from hippocampal tissue samples using enzymatic and mechanical dissociations. The treatment with Ara-C inhibits the growth of non-neuronal cells, facilitating the selective growth of hippocampal neurons.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Dissection, Dissociation, and Culture of Hippocampal Neurons from Pregnant Mouse/Rat

1. Dissection and dissociation of hippocampal neurons for culture
   1. Thaw protease enzyme solution (3 mg/mL protease 23 in Slice dissection solution, SLDS, Table 1) and pre-warm plating media (Table 2) at 37 °C.
   2. Dissect the hippocampus and rinse with SLDS.
      NOTE: Four hippocampi provide enough cells for one 2....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
12 mm coverslips	Warner Instruments	64-0702	For 24-well plate
25 mm coverslips	Fisher Scientific	12-545-102	For 6-well plate
Acetic acid	Fisher Scientific	A38-212
Poly-D-lysine	Sigma	P6407
Rat tail collagen	Roche	11 179 179 001
10X PBS	National Diagnostics	CL-253
Na2SO4	Fisher Scientific	S373-500
K2SO4	Fisher Scientific	P304-500
HEPES	Fisher Scientific	BP410-500
D-glucose	Fisher Scientific	D16-500
MgCl2	Fisher Scientific	BP214-500
NaOH	Fisher Scientific	SS255-1
Protease enzyme	Sigma	P4032
FBS	Gibco	26140
Sodium pyruvate	Gibco	11360-070
L-glutamine	Gibco	25030081
Penicillin/Streptomycin 100x (P/S)	Gibco	15140122
MEM Earle's Salts	Gibco	11090
B27 supplement	Gibco	17504-044
Neurobasal	Gibco	21103-049
Arabinosylcytosine (Ara-C)	Sigma	147-94-4
Opti-MEM media	Gibco	31985-070