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 Bovine Serum [FBS], 20 mM KCl, 25 mM NaHCO3, 30 mM D-glucose, 2 mM L-glutamine, 100 U/ml penicillin, 100 &#181;g/ml streptomycin and 6 &#181;g/ml ampicillin) and filter sterilize. Store at 4 &#176;C for months.</li>
	<li>Prepare 24 well culture plates containing 14 mm, number 1 thickness coverslips. Autoclave the coverslips and coat in 100 mg/L poly-d-lysine (PDL) as previously described 3. UV light sterilize O/N.</li>
</ol>
2. Preparation of CGC Culture Solutions
<ol>
	<li>Prepare &#8216;solution A&#8217; (100 ml) in autoclaved, UV light treated, sterile dH2O containing 250 mg of glucose, 300 mg of Bovine Serum Albumin [BSA], 955 mg of Phosphate Buffered Saline [PBS] (Ca2+ and Mg+ free), and 1 ml of 3.8% MgSO4&#183;7H2O.</li>
	<li>Prepare &#8216;solution B&#8217; (10 ml) containing 1 ml of 5 mg/ml trypsin diluted in 9 ml of solution A.</li>
	<li>Prepare &#8216;solution C&#8217; (10 ml) containing 1,000 units DNase, 0.5 mg of soybean trypsin inhibitor, 100 &#181;l of 3.8% MgSO4&#183;7H2O, diluted to 10 ml&#160;with solution A.</li>
	<li>Prepare &#8216;solution D&#8217; (20 ml) containing 3.2 ml of solution C, diluted to 20 ml with solution A.</li>
	<li>Prepare BSA/Earle&#8217;s Balanced Salt Solution (EBSS) solution containing EBSS, 25 mM NaHCO3, 3 mM MgSO4&#183;7H2O, and 4% BSA, pH 7.4, as previously described3.</li>
	<li>Prepare LME by adding pure LME into 5 ml of MEM culture medium to give a final concentration of 150 mM LME, return the solution to pH 7.4 using a benchtop pH meter, and filter sterilize using a 28 mm polyether sulfone (PES) 0.2 &#181;m syringe filter.</li>
</ol>
3. Culturing the CGCs
<ol>
	<li>Collect cerebella from 4-7 day old rat pups as previously described3. Place immediately in Petri dish containing 5 ml solution A on ice.</li>
	<li>Pour off excess solution from cerebella and place tissue in the Petri dish lid.</li>
	<li>Chop tissue finely with a well-flamed razor blade in at least three different directions.</li>
	<li>Add chopped tissue to solution B (rinse Petri dish with solution to ensure limited tissue is lost) and place in water bath at 37 &#176;C for 5 min. Shake gently every couple of min.</li>
	<li>Add solution D (20 ml) to the tube to neutralize the trypsin, shake and centrifuge at 65 x g for 5 min.</li>
	<li>Pour off supernatant.</li>
	<li>Resuspend in 4 ml of solution C. Triturate well (approximately 10 times each) with three flamed glass pipettes of decreasing diameter, until the suspension is as homogenous as possible.</li>
	<li>Slowly and gently add a few drops of this homogenate on top of the BSA/EBSS. If it starts to sink through the BSA/EBSS, triturate more and/or add a little more solution C. The homogenate should sit in a layer on top of the BSA/EBSS. Do not shake. Spin at 100 x g for 5 min.</li>
	<li>Remove supernatant and resuspend pellet (soft) in 1 ml of MEM medium.</li>
	<li>Count cells using a haemocytometer and plate at 800,000 cells/coverslip in 500 &#181;l of MEM medium. Place in an incubator at 37 &#176;C with 6% CO2.</li>
	<li>Change the medium the following day (24-36 hr after the prep). Make a solution of MEM medium containing 10 &#181;M of the cell cycle inhibitor arabinofuranosyl cytidine (AraC).
	<ol>
		<li>For each coverslip, retain 250 &#181;l of the old medium in a fresh 24 well plate, aspirate the rest, add 250 &#181;l of normal MEM medium and aspirate this to wash the cells, then add the 250 &#181;l of old medium back to the cells and top up with 250 &#181;l of AraC-containing medium. 
		Note: Cells can be used from 6 days in vitro (DIV).</li>
	</ol>
	</li>
</ol>
4. Selectively Depleting the Microglia (Performed at 6 DIV)
<ol>
	<li>Add pure LME to 5 ml of a separate aliquot of MEM medium to give a final concentration of 150 mM LME.</li>
	<li>Return the solution to pH 7.4 and filter sterilize using a 28 mm polyether sulfone (PES) 0.2 &#181;m syringe filter.</li>
	<li>Dilute solution to a concentration of 50 mM (2 x LME) in MEM medium and place in a water bath at 37 oC for 10 min along with normal MEM medium for control cultures.</li>
	<li>Remove half (250 &#181;l) the MEM medium from CGC cultures and retain at 37 &#176;C.</li>
	<li>Treat cells with MEM medium containing 2 x LME (250 &#181;l), i.e. diluted 1:1 giving a well concentration of 25 mM, or with pre-warmed MEM medium without LME (250 &#181;l) for control cultures.</li>
	<li>Incubate cells at 37 &#176;C in a humidified atmosphere with 6% CO2 for 1 hr.</li>
	<li>Wash cells twice in fresh pre-warmed MEM medium to remove LME-containing media, and then replace the retained culture medium and an equal volume of fresh pre-warmed MEM medium to the corresponding wells.</li>
	<li>Return cultures to the incubator (humidified with 6% CO2 at 37 &#176;C) and leave to rest for 24 hr before any further treatment.</li>
</ol>

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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. 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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

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Neuroscience

8.6K Views.  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. 