selectively removing microglia from a rat cerebellar granule neuron culture

Selectively Removing Microglia from a Rat Cerebellar Granule Neuron Culture

Begin this procedure by collecting the cerebella from four to seven-day-old rat pups. Place them immediately in a Petri dish containing five milliliters of solution A on ice. Then, remove excess solution from the cerebella and place the tissue in the Petri dish lid. Next, chop the tissue finely with a well-flamed razor blade in at least three different directions.
Subsequently, add the chopped tissue to solution B. Place it in a water bath at 37 degrees Celsius for five minutes, and shake it gently every couple of minutes. Then, add 20 milliliters of solution D to the tube to neutralize the trypsin. Shake and centrifuge at 65 g for five minutes. Afterward, pour off the supernatant. Resuspend it in four milliliters of solution C.
Triturate the sample 10 times with each of the three flamed glass pipettes of decreasing diameter until the suspension is as homogeneous as possible. Next, 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 add a little more solution C. The homogenate should sit in a layer on top of the BSA-EBSS.
Spin it at 100 g for five minutes, and do not shake. Then, remove the supernatant and resuspend the pellet in one milliliter of MEM medium. Count the cells using a hemocytometer, and plate them at 800,000 cells per coverslip in 500 microliters of MEM medium. After that, place them in an incubator at 37 degrees Celsius with 6% carbon dioxide. Then, make a solution of MEM medium containing 10 micromolar of the cell cycle inhibitor, AraC.
For each coverslip, retain 250 microliters of the old medium in a fresh 24-well plate and aspirate the rest. Add 250 microliters of normal MEM medium to wash the cells. Subsequently, add 250 microliters of old medium back to the cells, and top up with 250 microliters of AraC-containing medium.
In this procedure, add pure LME to five milliliters of MEM medium to give a final concentration of 150 millimolar LME. Return the solution to pH 7.4 using acid-base solution. Then, sterile filter it using a 28-millimeter PES with 0.2-micrometer syringe filter.
Next, dilute the LME solution to a concentration of 50 millimolar in MEM medium. Then, place it in a water bath at 37 degrees Celsius for 10 minutes, along with normal MEM medium for control cultures. After 10 minutes, remove 250 microliters of the MEM medium from each CGC culture. Then, retain the media at 37 degrees Celsius.
Next, treat the cells with MEM medium containing two times LME or with pre-warmed MEM medium without LME for control cultures. Incubate the cells at 37 degrees Celsius in a humidified atmosphere with 6% carbon dioxide for one hour. Afterward, wash the cells twice in fresh pre-warmed MEM medium to remove the LME-containing media.
Then, replace the retained culture medium with an equal amount of fresh pre-warmed MEM medium. Lastly, incubate the cultures in 6% humidified carbon dioxide at 37 degrees Celsius.

selectively-removing-microglia-from-rat-cerebellar-granule-neuron

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Neuronal Culture Techniques

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
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% Fetal Bovine Serum [FBS], 20 mM KCl, 25 mM NaHCO3, 30 mM D-glucose, 2 mM L-glutamine, 100 U/ml penicillin, 100 &#956;g/ml streptomycin and 6 &#956;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). UV light sterilize O/N.</li>
</ol>
2. Preparation of Cerebellar Granule Cell (CGC) Culture Solutions
<ol>
	<li>Prepare &#39;solution A&#39; (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 &#39;solution B&#39; (10 ml) containing 1 ml of 5 mg/ml trypsin diluted in 9 ml of solution A.</li>
	<li>Prepare &#39;solution C&#39; (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 with solution A.</li>
	<li>Prepare &#39;solution D&#39; (20 ml) containing 3.2 ml of solution C, diluted to 20 ml with solution A.</li>
	<li>Prepare BSA/Earle&#39;s Balanced Salt Solution (EBSS) solution containing EBSS, 25 mM NaHCO3, 3 mM MgSO4&#183;7H2O, and 4% BSA, pH 7.4.</li>
	<li>Prepare L-leucine Methyl Ester (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. Place immediately in Petri dish containing 5 ml solution A on ice.</li>
	<li>Pour off the excess solution from the cerebella and place the 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 a solution to ensure limited tissue is lost) and place in the 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 the supernatant and resuspend the pellet (soft) in 1 ml of MEM medium.</li>
	<li>Count cells using a hemocytometer 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>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Forceps</td>
			<td>Sigma-Aldrich</td>
			<td>F4142</td>
			<td>The curved end facilitates removal of the cerebellum</td>
		</tr>
		<tr>
			<td>Micro-dissecting scissors</td>
			<td>Sigma-Aldrich</td>
			<td>S3146</td>
			<td>Straight, sharp point facilitates rodent P4-7 dissection</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</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</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</td>
			<td>Sigma-Aldrich</td>
			<td>T6414</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Jebelli, J., et al. <a href="https://app.jove.com/v/52983">Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester.</a> 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.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of Instruments, ...

Take a harvested rat cerebellum and chop it into fragments.
Transfer the fragments to a tube containing trypsin and incubate.
Trypsin digests the tissue&#39;s extracellular matrix, loosening the cerebellar granule neurons, as well as non-neuronal cells such as microglia and astrocytes.
Add a buffer containing a trypsin inhibitor and DNase. The inhibitor inactivates trypsin while DNase degrades contaminating DNA.
Centrifuge and discard the supernatant.
Resuspend the tissue in a buffer containing the inhibitor and DNase, and mechanically dissociate it to form a single-cell suspension.
Overlay the suspension onto a protein-containing buffer solution.
Centrifuge and discard the supernatant containing cellular debris.
Resuspend the cells in media and seed them on poly-L-lysine-coated coverslips to facilitate cellular attachment.
Remove the media. Wash the cells with fresh media, and then, add media containing a cell cycle inhibitor to prevent glial cell proliferation.
Replace half of the media with fresh media containing a lysosome inhibitor.
The inhibitor selectively targets microglia and disrupts lysosomal membranes, causing microglial death and removal from the culture.

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视频: 从大鼠小脑颗粒神经元培养物中选择性去除小胶质细胞 - 实验

A Simplified Method for Ultra-Low Density, Long-Term Primary Hippocampal Neuron Culture

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic hippocampal neurons can often grow successfully on glass coverslips at high density under serum-free conditions, but low density cultures typically require a supply of trophic factors by co-culturing them with a glia feeder layer, preparation of which can be time-consuming and laborious. In addition, the presence of glia may confound interpretation of results and preclude studies on neuron-specific mechanisms. Here, a simplified method is presented for ultra-low density (~2,000 neurons/cm2), long-term (&#62;3 months) primary hippocampal neuron culture that is under serum free conditions and without glia cell support. Low density neurons are grown on poly-D-lysine coated coverslips, and flipped on high density neurons grown in a 24-well plate. Instead of using paraffin dots to create a space between the two neuronal layers, the experimenters can simply etch the plastic bottom of the well, on which the high density neurons reside, to create a microspace conducive to low density neuron growth. The co-culture can be easily maintained for &#62;3 months without significant loss of low density neurons, thus facilitating the morphological and physiological study of these neurons. To illustrate this successful culture condition, data are provided to show profuse synapse formation in low density cells after prolonged culture. This co-culture system also facilitates the survival of sparse individual neurons grown in islands of poly-D-lysine substrates and thus the formation of autaptic connections.

Low density cultures of primary hippocampal neurons usually require glia feeder layer to supply neurotrophic factors and sustain longevity. We describe here a simplified method to culture ultra-low density neurons on glass coverslips in the presence of a high density neuronal feeder layer, which facilitates investigation of specific neuronal-autonomous mechanisms.

的简化方法超低密度，长期主要海马神经元文化

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic ...

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神经科学

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

间接神经元的星形胶质细胞共培养试验:一个<em&gt;体外</em&gt;设置 - 为神经元的神经胶质细胞相互作用的详细调查

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

Presynapse Formation Assay Using Presynapse Organizer Beads and &ldquo;Neuron Ball&rdquo; Culture

During neuronal development, synapse formation is an important step to establish neural circuits. To form synapses, synaptic proteins must be supplied in appropriate order by transport from cell bodies and/or local translation in immature synapses. However, it is not fully understood how synaptic proteins accumulate in synapses in proper order. Here, we present a novel method to analyze presynaptic formation by using the combination of neuron ball culture with beads to induce presynapse formation. Neuron balls that is neuronal cell aggregates provide axonal sheets far from cell bodies and dendrites, so that weak fluorescent signals of presynapses can be detected by avoiding overwhelming signals of cell bodies. As beads to trigger presynapse formation, we use beads conjugated with leucine-rich repeat transmembrane neuronal 2 (LRRTM2), an excitatory presynaptic organizer. Using this method, we demonstrated that vesicular glutamate transporter 1 (vGlut1), a synaptic vesicle protein, accumulated in presynapses faster than Munc18-1, an active zone protein. Munc18-1 accumulated translation-dependently in presynapse even after removing cell bodies. This finding indicates the Munc18-1 accumulation by local translation in axons, not transport from cell bodies. In conclusion, this method is suitable to analyze accumulation of synaptic proteins in presynapses and source of synaptic proteins. As neuron ball culture is simple and it is not necessary to use special apparatus, this method could be applicable to other experimental platforms.

Presynapse formation is a dynamic process including accumulation of synaptic proteins in proper order. In this method, presynapse formation is triggered by beads conjugated with a presynapse organizer protein on axonal sheets of &#8220;neuron ball&#8221; culture, so that accumulation of synaptic proteins is easy to be analyzed during presynapse formation.

使用预突子形成测定使用预突子组织者珠和"神经元球"文化

During neuronal development, synapse formation is an important step to establish neural circuits. To form synapses, synaptic proteins must be supplied in ...

Selectively Removing Microglia from a Rat Cerebellar Granule Neuron Culture

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Selectively Removing Microglia from a Rat Cerebellar Granule Neuron Culture