isolating and culturing murine cerebellar granular neuron progenitors

Isolating and Culturing Murine Cerebellar Granular Neuron Progenitors

Transfer three to five cerebella into 15-milliliter tubes. Wash cerebella with HBSS glucose before centrifugation. Centrifuge the tubes to collect the tissue. After the final wash, homogenize the cerebella by gently pipetting up and down two to three times with a 1-milliliter pipette until the fragments are 0.5 to 1 cubed millimeters in size.
Gently remove all liquid until 2.5 milliliters are left. Then, add 0.05% trypsin into the tube with HBSS glucose containing the cerebella, and incubate the tissue in a 37 degrees Celsius water bath for 15 minutes. Invert the tissue every 1 to 3 minutes. Following the incubation, stop the digestion by adding 5 milliliters of cGMP cell culture medium or CGM, and collect the tissue by centrifugation as before.
After centrifuging, remove the supernatant and add 1 milliliter CGM. Triturate the tissue with the 1-milliliter pipette tip, avoiding the formation of air bubbles. Then, add 5 milliliters of CGM, and incubate the mixture for two minutes on ice to settle tissue remnants. Once the tissue has settled, transfer the supernatant into a new 15-milliliter tube. Then, add 2 milliliters of CGM to the residual tissue, and repeat the trituration procedure before harvesting the supernatant and discarding the tissue remnants.
Pool the supernatants from each 15-milliliter tube of tissue, and centrifuge to collect the cerebellar cells. Resuspend the pellet in 10 milliliters of CGM. Since astrocytes adhere faster and stronger to poly-D-lysine than cGMPs, add up to 4 milliliters of cell suspension to the wells of poly-D-lysine-coated 6-well plates, and incubate for 20 minutes at 37 degrees Celsius to remove astrocytes.
Shake the plate. Collect the supernatant in a 15-milliliter tube, and then centrifuge the supernatant as before. Resuspend the pellet in 10 milliliters of CGM, and count the cells with a Neubauer counting chamber. After 4 to 6 hours, the adhered cGMPs appear round, and are proliferative. Seed the cells in CGM on poly-L-ornithine-coated plates, and incubate at 37 degrees Celsius, 5% CO2, and 100% relative humidity.

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1.&#160;Preparations
<ol>
	<li>Preparations for CGNP (cerebellar granular neuron progenitor) isolation
	<ol>
		<li>Organize an appropriate mating of mice to generate P5-P7 animals to isolate the cerebellum (3-5 animals per ChIP and condition).</li>
		<li>Prepare HBSS/Glucose by adding 6 mg/mL glucose to the HBSS buffer.</li>
		<li>Prepare the CGNP cell culture medium (CGM) by adding 1 % (v/v) N2 supplement, 1 % (v/v) Penicillin-Streptomycin-Neomycin, 25 mM KCl and 10 % FCS to DMEM-F12. For CGNP cultivation, prepare CGM medium without FCS but including 0.6 &#181;g/mL sonic hedgehog (SHH) (CGM-SHH) and equilibrate it to 37 &#176;C when needed.</li>
		<li>Coat 6-well cell culture plates with 100 &#181;g/mL poly-D-lysine for 1-2 h at RT for glia-cell removal. Afterwards wash twice with sterile ddH2O and let the plates dry. Store the plates for maximum one week at 4 &#176;C.</li>
		<li>For cultivation of CGNPs, coat 6-well cell culture plates with poly-L-ornithine (0.1 mg/mL) at 4 &#176;C overnight. Afterwards, wash twice with H2O for cell culture and let the plates dry. 
		NOTE: The plates can be stored for a maximum of one week at 4 &#176;C.</li>
		<li>Prepare 0.025 % trypsin (w/v) in HBSS/Glucose and equilibrate it to 37 &#176;C when needed.</li>
	</ol>
	</li>
	<li>Neural Progenitor Isolation of Brain Tissue
	<ol>
		<li>Isolation and optional cultivation of CGNPs (cerebellar granular neuron progenitors)
		<ol>
			<li>Euthanize P5-7 animals by decapitation using scissors. Remove the scalp skin, open the skull, and remove the brain using small scissors and forceps. Isolate the cerebellum and transfer it to ice-cold HBSS/Glucose. Remove all meninges and blood vessels and transfer the cerebella into 15 mL tubes filled with cold HBSS/Glucose.</li>
			<li>Wash the cerebella three times with 10 mL ice-cold HBSS/Glucose (collect them by centrifugation at 650 x g for 5 min at 4 &#176;C) and homogenize cerebella subsequently by gently pipetting up and down with a 1 mL pipette (maximum 2-3 times) to get 0.5-1 mm3 fragments.</li>
			<li>Add 5 mL of 0.025% trypsin in HBSS/Glucose and incubate the tissue under constant stirring in a 37 &#176;C water bath for 15 min. Stop the digestion by adding 5 mL of CGM and collect the tissue by centrifugation at 650 x g for 5 min at 4 &#176;C.</li>
			<li>Remove the supernatant and triturate the tissue with a 1 mL pipet tip in 1 mL CGM and transfer it to a new 15 mL tube. 
			NOTE: It is important to avoid air bubbles.
			<ol>
				<li>Add 5 mL CGM and incubate the mixture for 2 min on ice to settle tissue remnants. Transfer the supernatant into a new 15 mL tube. Add 2 mL CGM to the residual tissue and repeat the trituration procedure.</li>
			</ol>
			</li>
			<li>Pool the supernatants (without tissue remnants, about 10 mL in total) and centrifuge at 650 x g for 5 min at 4 &#176;C to collect the cerebellar cells. Resuspend the pellet in 10 mL CGM.</li>
			<li>Since astrocytes adhere faster and stronger to poly-D-lysine than CGNPs, plate the cells on 100 &#181;g/mL poly-D-lysine coated 6-well-plates (up to 4 mL per well) and incubate for 20 min at 37 &#176;C to remove the astrocytes.</li>
			<li>Shake the plate and collect the supernatant. Then centrifuge at 650 x g for 5 min at 4 &#176;C in a 15 mL tube. Resuspend the pellet in 10 mL CGM and count the cells with a Neubauer counting chamber. 
			NOTE: CGNPs are round, small, and show a halo when imaged using phase contrast microscopy.</li>
			<li>If required, seed the cells in 37 &#176;C pre-warmed CGM on poly-L-ornithine-coated plates (3 x 106 cells per 6-well) and incubate them at 37 &#176;C, 5% CO2 and 100% relative humidity. 
			NOTE: Leaving CGM on the plates (and with that FBS) will result in the differentiation of the CGNPs into cerebellar granular neurons (CGNs).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>B27 Supplement (50x)</td>
			<td>Life Technologies</td>
			<td>17504044</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CCM</td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>Life Technologies</td>
			<td>11320-033</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CGM</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum 10% (v/v)</td>
			<td>Gibco</td>
			<td>10082147</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CPC isolation and CGM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G5767</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CGNP isolation</td>
		</tr>
		<tr>
			<td>Glutathione (1.25 mg/ml)</td>
			<td>Sigma-Aldrich</td>
			<td>G4251</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CCM</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Carl Roth</td>
			<td>3187</td>
			<td>Category: ChIP 
			Abbreviation/Comment: For cell fixation</td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution</td>
			<td>Life Technologies</td>
			<td>14025-100</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: HBSS</td>
		</tr>
		<tr>
			<td>L-glutamine (200 mM)</td>
			<td>Life Technologies</td>
			<td>25030081</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CCM</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma-Aldrich</td>
			<td>L2020</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CPC culturing</td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Life Technologies</td>
			<td>17502048</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CGM</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CCM</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin-Neomycin 1% (v/v)</td>
			<td>Life Technologies</td>
			<td>15640055</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: PSN, for CCM and CGM</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Life Technologies</td>
			<td>10010023</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: PBS, for CPC isolation</td>
		</tr>
		<tr>
			<td>Poly-D-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P6407</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CGNP isolation</td>
		</tr>
		<tr>
			<td>Poly-L-ornithine hydrobromide</td>
			<td>Sigma Aldrich</td>
			<td>P3655</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CPC culturing</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Thermo Fisher</td>
			<td>AM9640G</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: KCl, for CGM</td>
		</tr>
		<tr>
			<td>Sonic hedgehock (SHH)</td>
			<td>Sigma-Aldrich</td>
			<td>SRP6004</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CGNP isolation</td>
		</tr>
		<tr>
			<td>Superoxide dismutase (1mg/ml)</td>
			<td>Sigma-Aldrich</td>
			<td>S7571</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CCM</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0,05% (w/v)</td>
			<td>Sigma Aldrich</td>
			<td>59417C</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CPC isolation</td>
		</tr>
	</tbody>
</table>

Source: Bovio, P., et al., <a href="https://app.jove.com/v/56631">Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark.</a> J. Vis. Exp. (2018)
This video demonstrates the isolation and cultivation of cerebellar neural progenitor cells from a mouse pup&#39;s cerebellum. Cerebellar granule neuron progenitors are isolated and purified through differential adhesion, effectively removing astrocytes. These cultured progenitors subsequently differentiate into cerebellar granule neurons.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1.&#160;Preparations

	Preparations ...

Take mouse pup cerebella.
Homogenize them into smaller fragments.
Add a proteolytic enzyme to digest the tissue&#8217;s extracellular matrix, loosening cerebellar granular progenitors, or CGNPs, and astrocytes.
Add CGNP culture media containing serum to stop the enzymatic activity. Centrifuge and discard the enzyme-rich supernatant.
Resuspend the tissue fragments in CGNP media. Mechanically dissociate the tissue to release the cells.
Once the debris settles, transfer the supernatant containing the cells to a fresh tube.
Centrifuge and remove the supernatant with debris.
Resuspend the cells in CGNP media, and seed them onto multi-well plate wells coated with poly-D-lysine. Incubate.
Astrocytes settle and adhere strongly to the poly-D-lysine coating, compared to CGNPs.
Shake the plate and collect the supernatant containing CGNPs. Centrifuge and remove the supernatant.
Resuspend CGNPs in CGNP media and seed them onto a poly-L-ornithine-coated multi-well plate. Incubate.
CGNPs attach to the coated surface and proliferate, aided by the media components and poly-L-ornithine.

14 조회수. Isolating and culturing murine Cerebellar Granular Neuron Progenitors(쥐 소뇌 과립 뉴런 전구 세포)

비디오: Isolating and culturing murine Cerebellar Granular Neuron Progenitors(쥐 소뇌 과립 뉴런 전구 세포) - 실험

Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the co-existence of glial cells and neurons in CGN culture might lead to biases in the accurate assessment of neuronal viability. Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining has been used to measure cell viability by simultaneously evaluating the viable and dead cells. We used FDA-PI double staining to improve the sensitivities of the colorimetric assays and to evaluate neuronal viability in CGNs. Furthermore, we added blue fluorescent DNA stains (e.g., Hoechst) to improve the accuracy. This protocol describes how to improve the accuracy of assessment of neuronal viability by using these methods in CGN culture. Using this protocol, the number of glial cells can be excluded by using fluorescence microscopy. A similar strategy can be applied to distinguish the unwanted glial cells from neurons in various mixed cell cultures, such as primary cortical culture and hippocampal culture.

This protocol describes how to accurately measure neuronal viability using Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining in cultured cerebellar granule neurons, a primary neuronal culture used as an in vitro model in neuroscience and neuropharmacology research.

소뇌 과립 신경 세포 배양에서 Fluorescein Diacetate-Propidium Iodide 이중 염색을 이용한 신경 생존력의 평가

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the ...

연구

JoVE Journal

신경과학

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

신경 죽음 및 마우스 기본 소 뇌과 립 신경에 변성을 모델링

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development

Neurons undergo dynamic changes in their structure and function during brain development to form appropriate connections with other cells. The rodent cerebellum is an ideal system to track the development and morphogenesis of a single cell type, the cerebellar granule neuron (CGN), across time. Here, in vivo electroporation of granule neuron progenitors in the developing mouse cerebellum was employed to sparsely label cells for subsequent morphological analyses. The efficacy of this technique is demonstrated in its ability to showcase key developmental stages of CGN maturation, with a specific focus on the formation of dendritic claws, which are specialized structures where these cells receive the majority of their synaptic inputs. In addition to providing snapshots of CGN synaptic structures&#160;throughout cerebellar development, this technique can be adapted to genetically manipulate granule neurons in a cell-autonomous manner to study the role of any gene of interest and its effect on CGN morphology, claw development, and synaptogenesis.

Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development

Here we describe a method to visualize synaptogenesis of granule neurons in the mouse cerebellum over the time course of postnatal brain development when these cells refine their synaptic structures and form synapses to integrate themselves into the overall brain circuit.

소뇌 과립 뉴런 형태 및 시냅스 발달을 연구하기 위해 생체 내 출생 후 전기천공법 활용

Neurons undergo dynamic changes in their structure and function during brain development to form appropriate connections with other cells. The rodent ...

Isolating and Culturing Murine Cerebellar Granular Neuron Progenitors

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Isolating and Culturing Murine Cerebellar Granular Neuron Progenitors