Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester

Summary

Microglia can influence neurons and other glia in culture by various non-cell autonomous mechanisms. Here, we present a protocol to selectively deplete microglia from primary neuronal cultures. This method has the potential to elucidate the role of microglial-neuronal interactions, with implications for neurodegenerative conditions where neuroinflammation is a hallmark feature.

Abstract

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating innate immunity. Primary rodent cell culture models have greatly advanced our understanding of neuronal-glial interactions, but only recently have methods to specifically eliminate microglia from mixed cultures been utilized. One such technique – described here – is the use of L-leucine methyl ester, a lysomotropic agent that is internalized by macrophages and microglia, wherein it causes lysosomal disruption and subsequent apoptosis^13,14. Experiments using L-leucine methyl ester have the power to identify the contribution of microglia to the surrounding cellular environment under diverse culture conditions. Using a protocol optimized in our laboratory, we describe how to eliminate microglia from P5 rodent cerebellar granule cell culture. This approach allows one to assess the relative impact of microglia on experimental data, as well as determine whether microglia are playing a neuroprotective or neurotoxic role in culture models of neurological conditions, such as stroke, Alzheimer’s or Parkinson’s disease.

Introduction

The human brain comprises an estimated 85 billion neurons and a further 85 billion non-neuronal cells including glia¹. For the greater part of the past 100 years neuroscientists have focused predominantly on the neuronal cell population, believing glial cells to be little more than passive support cells that provided structural support for the neurons – hence the Greek etymology of ‘glia’ translated to English as ‘glue’. Recently, however, it has become increasingly evident that neuronal-glial interactions may be far more fundamental to basic aspects of neurobiology, neurophysiology, and the genesis and progression of many neur....

Protocol

All experiments described herein were performed in accordance with the United Kingdom Animals (Scientific Procedures) Act of 1986.

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% Foetal 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....

Results

The ability of this technique to selectively eliminate microglia from CGC and/or mixed cultures relies upon the subsequent ability of the investigator to accurately identify and differentiate microglia from their surrounding cells. This can be achieved using a microglial-specific cell maker, such as isolectin-B4, as illustrated in Figure 1. As demonstrated in Figure 2, no observable changes to astrocyte and neuronal density and morphology were recorded with respect to each main treatment.......

Discussion

The most important steps to ensure the successful selective elimination of microglia from CGC and/or mixed cultures are: 1) maintaining a sterile and healthy CGC culture; 2) filter sterilizing the LME-containing medium and returning the solution to pH 7.4; 3) keeping the retained CGC media and LME-containing media at 37 °C to avoid heat shock; and 4) working quickly to reduce the time cells are kept outside the incubator.

We used 25 mM LME to deplete microglia from our CGC cult.......

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
Mouse anti-ED1 antibody	Abcam	ab31630