This research was support by an Aims2Cure, UK and a UCL Impact Award Ph.D. studentship to JMP and an MRC Capacity Building Ph.D. studentship in Dementia to JMP.

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
<ol>
	<li>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.</li>
	<li>Make 500 ml of minimum essential media (MEM) culture medium (10% Foetal B...

<ol>
	<li>Herculano-Houzel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+remarkable,+yet+not+extraordinary,+human+brain+as+a+scaled-up+primate+brain+and+its+associated+cost.">The remarkable, yet not extraordinary, human brain as a scaled-up primate brain and its associated cost.</a> PNAS. 26 (109), 10661-10668 (2012).</li><li>Evans, G. J., &amp;Pocock, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+neurotransmitter+release+by+dihydropyridine-sensitive+calcium+channels+involves+tyrosine+phosphorylation.">Modulation of neurotransmitter release by dihydropyridine-sensitive calcium channels involves tyrosine phosphorylation.</a> Eur J Neuro. 11 (1), 279-292 (1999).</li><li>Kingham, P. J., Cuzner, M. L., Pocock, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptotic+pathways+mobilized+in+microglia+and+neurones+as+a+consequence+of+chromogranin+A-induced+microglial+activation.">Apoptotic pathways mobilized in microglia and neurones as a consequence of chromogranin A-induced microglial activation.</a> J Neurochem. 73 (2), 538-547 (1999).</li><li>Contestabile, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebellar+granule+cells+as+a+model+to+study+mechanisms+of+neuronal+apoptosis+or+survival+in+vivo+and+in+vitro.">Cerebellar granule cells as a model to study mechanisms of neuronal apoptosis or survival in vivo and in vitro.</a> Cerebellum. 1 (1), 41-55 (2002).</li><li>Kramer, D., Minichiello, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture+of+primary+cerebellar+granule+cells.">Cell culture of primary cerebellar granule cells.</a> Methods Mol Biol. 633, 233-239 (2010).</li><li>Facci, L., Skaper, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+rat+cerebellar+granule+neurons+and+application+to+identify+neuroprotective+agents.">Culture of rat cerebellar granule neurons and application to identify neuroprotective agents.</a> Methods Mol Biol. 846, 23-37 (2012).</li><li>Thiele, D. L., Kurosaka, M., Lipsky, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotype+of+the+accessory+cell+necessary+for+mitogen-stimulated+T+and+B+cell+responses+in+human+peripheral+blood:+delineation+by+its+sensitivity+to+the+lysosomotropic+agent,+L-leucine+methyl+ester.">Phenotype of the accessory cell necessary for mitogen-stimulated T and B cell responses in human peripheral blood: delineation by its sensitivity to the lysosomotropic agent, L-leucine methyl ester.</a> J Immunology. 131 (5), 2282-2290 (1983).</li><li>Giulian, D., Vaca, K., Corpuz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+glia+release+factors+with+opposing+actions+upon+neuronal+survival.">Brain glia release factors with opposing actions upon neuronal survival.</a> J Neurosci. 13 (1), 29-37 (1993).</li><li>Guillemin, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obtention+and+characterization+of+primary+astrocyte+and+microglial+cultures+from+adult+monkey+brains.">Obtention and characterization of primary astrocyte and microglial cultures from adult monkey brains.</a> J Neurosci Res. 49 (5), 576-591 (1997).</li><li>Hewett, J. A., Hewett SJ, ., Winkler, S., Pfeiffer SE, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+nitric+oxide+synthase+expression+in+cultures+enriched+for+mature+oligodendrocytes+is+due+to+microglia.">Inducible nitric oxide synthase expression in cultures enriched for mature oligodendrocytes is due to microglia.</a> J Neurosci Res. 56 (2), 189-198 (1999).</li><li>Jebelli, J., Hooper, C., &amp;Pocock, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+p53+activation+is+detrimental+to+neuronal+sysnapses+during+activaton-induced+inflammation:+implications+for+neurodegeneration.">Microglial p53 activation is detrimental to neuronal sysnapses during activaton-induced inflammation: implications for neurodegeneration.</a> Neurosci Lett. 7 (583), 92-97 (2014).</li><li>Frade, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+induces+release+of+glutathione+from+cultured+rats+astrocytes+&#8211;+a+possible+neuroprotective+mechanism.">Glutamate induces release of glutathione from cultured rats astrocytes &#8211; a possible neuroprotective mechanism.</a> J Neurochem. 105 (4), 1144-1152 (2008).</li><li>Hamby, M. E., Uliasz, T. F., Hewett, S. J., Hewett, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+an+improved+procedure+for+the+removal+of+microglia+from+confluent+monolayers+of+primary+astrocytes.">Characterization of an improved procedure for the removal of microglia from confluent monolayers of primary astrocytes.</a> J Neurosci Methods. 150 (1), 128-137 (2006).</li><li>Morgan, S. C., Taylor, D. L., Pocock, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+release+activators+of+neuronal+proliferation+mediated+by+activation+of+mitogen-activated+protein+kinase,+phosphatidylinositol-3-kinase/Akt+and+delta-Notch+signalling+cascades.">Microglia release activators of neuronal proliferation mediated by activation of mitogen-activated protein kinase, phosphatidylinositol-3-kinase/Akt and delta-Notch signalling cascades.</a> J Neurochem. 90 (1), 89-101 (2004).</li><li>Crocker, S. J., Frausto, R. F., Whitton, J. L., Milner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+to+establish+microglia-free+astrocyte+cultures:+comparison+of+matrix+metalloproteinase+expression+profiles+in+pure+cultures+of+astrocytes+and+microglia.">A novel method to establish microglia-free astrocyte cultures: comparison of matrix metalloproteinase expression profiles in pure cultures of astrocytes and microglia.</a> Glia. 56 (11), 1187-1198 (2008).</li><li>Kumamaru, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomal+clodronate+selectively+eliminates+microglia+from+primary+astrocyte+cultures.">Liposomal clodronate selectively eliminates microglia from primary astrocyte cultures.</a> J Neuroinflamm. 9, 116 (2012).</li></ol>

The authors declare that they have no competing financial interests.

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 &#176;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...

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 &#8211; hence the Greek etymology of &#8216;glia&#8217; translated to English as &#8216;glue&#8217;. 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 neurodegenerative diseases. Cerebellar granule cells (CGCs), the most abundant homogenous neuronal population in the human brain, dominate the cerebellum and make up more than 90% of its cellular constituents. Consequently, these cells have been used extensively in vitro as a model system for the study of neuronal development, function, and pathology2-6.
However, CGC cultures still contain microglia and other glia in arguably significant proportions. As a result, CGC data putatively displaying direct neuronal responses to different cell treatments may in fact arise &#8211; in part or in total &#8211; from the indirect secondary response of neighbouring glia in the culture. To assess this, we selectively eliminated microglial from CGC neuronal cultures with the aid of L-leucine methyl ester (LME). LME is a lysomotropic agent originally used to selectively destroy macrophages7, and has since been used to also selectively deplete microglia from neural, astrocyte, and mixed glial cultures8,9,10. LME is internalized by macrophages and microglia, wherein it causes lysosomal disruption and subsequent apoptosis13,14. Macrophages and microglia are characteristically rich in lysosomes, causing them to be particularly vulnerable upon exposure to LME treatment. This protocol provides a powerful, yet simple and easy way to ascertain the contribution of microglia in experiments utilizing CGC and other neuronal/glial culture systems.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Forceps&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>F4142</td>
			<td>The curved end facilitates removal of the cerebellum&#160;</td>
		</tr>
		<tr>
			<td>Micro-dissecting scissors</td>
			<td>Sigma-Aldrich</td>
			<td>S3146</td>
			<td>Straight, sharp point facilitates rodent P4-7 dissection&#160;</td>
		</tr>
		<tr>
			<td>L-leucine methyl ester hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>7517-19-3</td>
			<td></td>
		</tr>
		<tr>
			<td>EBSS solution&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>E7510-500 ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>27964-99-4</td>
			<td>Coat coverslips 1 day before use&#160;</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>Sigma-Aldrich</td>
			<td>D5025</td>
			<td></td>
		</tr>
		<tr>
			<td>Soybean trypsin inhibitor&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T6414</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-ED1 antibody&#160;</td>
			<td>Abcam</td>
			<td>ab31630</td>
			<td></td>
		</tr>
	</tbody>
</table>

selective depletion of microglia from cerebellar granule cell cultures using l-leucine methyl ester

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 &#8211; described here &#8211; 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 apoptosis13,14. 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&#8217;s or Parkinson&#8217;s disease.

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...

Watch this Scientific Journal Video about Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester at JoVE.com

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

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.

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating ...

selective-depletion-microglia-from-cerebellar-granule-cell-cultures

Research

JoVE Journal

Neuroscience

8.6K Görüntüleme.  University of Washington. 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. 