Selectively Removing Microglia from a Rat Cerebellar Granule Neuron Culture

Abstract

Source: Jebelli, J., et al. Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester. J. Vis. Exp. (2015)

This video demonstrates a technique for the selective removal of microglia from a primary rat neuronal culture. Cerebellar granule neurons are treated with an antimetabolite to eliminate proliferating glial cells. Additionally, they are treated with lysosome-targeting agents to deplete microglia from the culture.

Protocol

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

1. Preparation of Instruments, Culture Media, and Dishes

1. Prepare two stainless steel laboratory dissecting scissors and two stainless steel laboratory forceps. Autoclave all instruments and place in a culture hood O/N under ultraviolet light (UV) light for sterilization.
2. Make 500 ml of minimum essential media (MEM) culture medium (10% Fetal Bovine Serum [FBS], 20 mM KCl, 25 mM NaHCO₃, 30 mM D-glucose, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 6 μ....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Forceps	Sigma-Aldrich	F4142	The curved end facilitates removal of the cerebellum
Micro-dissecting scissors	Sigma-Aldrich	S3146	Straight, sharp point facilitates rodent P4-7 dissection
L-leucine methyl ester hydrochloride	Sigma-Aldrich	7517-19-3
EBSS solution	Sigma-Aldrich	E7510-500 ml
Poly-D-lysine	Sigma-Aldrich	27964-99-4	Coat coverslips 1 day before use
Bovine serum albumin (BSA)	Sigma-Aldrich	A9418
Phosphate buffered saline (PBS)	Sigma-Aldrich	P4417
DNase	Sigma-Aldrich	D5025
Soybean trypsin inhibitor	Sigma-Aldrich	T6414