Name: 视频: 小鼠原代小脑颗粒神经元的分离和培养 - 概念
Uploaded: 2024-07-31T15:22:08.000+00:00
Duration: 1 min 18 s
Description: 161 次观看. 来源:Laaper， M. 等人 小鼠原代小脑颗粒神经元神经元死亡和变性建模。J. Vis. Exp. （2017 年） 该视频演示了分离和培养原代小鼠小脑颗粒神经元的技术。用抗代谢物处理细胞以消除增殖的神经胶质细胞并产生小脑颗粒神经元的纯培养物。

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Experimental Preparation
NOTE: The following stock solutions can be prepared and maintained until use.
<ol>
	<li>Dissection Solution&#8203;
	<ol>
		<li>Dissolve 3.62 g of sodium chloride (NaCl), 0.2 g of potassium chloride (KCl), 0.069 g of sodium phosphate (NaH2PO4&#8729; H2O), and 1.306 g of D-(+)-Glucose in 500 mL of distilled H2O. Then add 12.5 mL of HEPES Buffer, 450 &#181;L of Phenol red solution, and 20 mL of BSA fraction V to the solution. Adjust to pH 7.4 using 1 N sodium hydroxide (NaOH).</li>
		<li>Sterilize with a 0.22 &#181;m filter and store at 4 &#176;C. This solution will remain stable for up to one week to 10 days.</li>
	</ol>
	</li>
	<li>Trypsin
	<ol>
		<li>Prepare a stock solution at a concentration of 25 g/L of trypsin in 0.9% sodium chloride. Store the solution at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Trypsin Inhibitor
	<ol>
		<li>Prepare a stock with a concentration of 10 mg/mL by dissolving 1 g of chicken egg white trypsin inhibitor in 100 mL of 1 mM HCl. Store this solution at -20 &#176;C.</li>
	</ol>
	</li>
	<li>DNase&#8203;
	<ol>
		<li>Dissolve 100 mg of DNase 1 in 10 mL of sterile water to generate a 10 mg/mL stock. Store the solution at -20 &#176;C.</li>
	</ol>
	</li>
	<li>MgSO4
	<ol>
		<li>Add 6.02 g of magnesium sulfate (MgSO4) to 50 mL of distilled water (H2O) to generate a 1 M stock. Store the solution at 4 &#176;C.</li>
	</ol>
	</li>
	<li>CaCl2
	<ol>
		<li>Dissolve 0.735 g of calcium chloride (CaCl2&#8729; 2H2O) in 50 mL of distilled H2O to a stock concentration of 100 mM. Store solution at 4 &#176;C.</li>
	</ol>
	</li>
	<li>KCl&#8203;
	<ol>
		<li>Dissolve 3.7275 g of KCl in 50 mL of distilled H2O to a stock concentration of 1 M. Store solution at 4 &#176;C.</li>
	</ol>
	</li>
	<li>D-(+)-Glucose
	<ol>
		<li>Dissolve 1.802 g of D-(+)-glucose in 50 mL of distilled H2O to a stock concentration of 100 mM. Store the solution at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Cell Culture Media
	<ol>
		<li>Prepare cell culture media as follows: 5 mL of heat-inactivated dialyzed Fetal Bovine Serum, 500 &#181;L of 200 mM L-Glutamine, 100 &#181;L of 50 mg/mL Gentamycin and 1 mL of 1.0 M KCl should be added to 45 mL of filter sterilized Eagle&#39;s minimal essential media (E-MEM) that is supplemented with 1.125 g/L D-Glucose to generate 50 mL of cerebellar granule neuron culture media. Store media at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Cytosine Beta-D- Arabino Furanoside&#8203;
	<ol>
		<li>Dissolve 48 mg of cytosine beta-D-arabinofuranoside in 10 mL of growth media to a stock concentration of 20 mM. Store solution at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Poly-D-Lysine
	<ol>
		<li>To generate a 2 mg/mL poly D-lysine stock, add 10 mL of sterile H2O to 20 mg of poly D-lysine. Stock poly D-lysine should be stored at -80 &#176;C in 100 &#181;L aliquots. 
		NOTE: The following solutions should be prepared prior to dissection and maintained at 4 &#176;C until use.</li>
	</ol>
	</li>
	<li>MgSO4 Supplemented Dissection Solution
	<ol>
		<li>Add 300 &#181;L of stock MgSO4 to 250 mL of dissection solution.</li>
	</ol>
	</li>
	<li>Trypsin Dissection Solution
	<ol>
		<li>Add 100 &#181;L of stock trypsin and 12 &#181;L of stock MgSO4 to 10 mL of dissection solution.</li>
	</ol>
	</li>
	<li>Trypsin Inhibitor Solution 1&#8203;
	<ol>
		<li>Add 164 &#181;L of stock trypsin inhibitor, 250 &#181;L of stock DNase 1 and 12 &#181;L of stock MgSO4 to 10 mL of dissection solution.</li>
	</ol>
	</li>
	<li>Trypsin Inhibitor Solution 2
	<ol>
		<li>Add 1040 &#181;L of stock trypsin inhibitor, 750 &#181;L of stock DNase 1 and 12 &#181;L of stock MgSO4 to 10 mL of dissection solution.</li>
	</ol>
	</li>
	<li>CaCl2 supplemented dissection solution
	<ol>
		<li>Add 10 &#181;L of stock CaCl2 and 25 &#181;L of stock MgSO4 to 10 mL of dissection solution.</li>
	</ol>
	</li>
	<li>Poly D-Lysine Coated Culture Dishes&#8203;
	<ol>
		<li>To coat culture dishes, dilute 100 &#181;L of stock poly D-lysine in 40 mL of sterile H2O. For 35 mm Nunc culture dishes, add 2 mL of diluted poly D-Lysine solution. For 4 well plates, add 300 &#181;L of diluted poly D-Lysine to each well.</li>
		<li>Let the poly D-lysine sit for 1 h before aspirating and washing each well with 4 mL or 600 &#181;L (for 35 mm culture dishes or 4 well plates, respectively) of sterile H2O. Then aspirate the H2O and let the dishes air dry for 1 h.</li>
	</ol>
	</li>
</ol>
2. Brain Extraction and Isolation of Cerebellum
<ol>
	<li>Decapitate a 6-7 day-old mouse pup using decapitation scissors. Perform the dissection of the brain in a sterile tissue culture hood.</li>
	<li>To remove the brain, grasp the head using a pair of forceps and cut through the skin toward the anterior of the head using microdissection scissors, as demonstrated in Figure 1. Then cut through the skull bone using a new pair of scissors to minimize the risk of contamination. Remove the skull covering the brain using forceps and tease out the brain using forceps or a spatula.&#8203;
	<ol>
		<li>As needed, cut through the optic nerve to facilitate getting the brain out as a whole.</li>
	</ol>
	</li>
	<li>Place the brain in the dissection solution which is supplemented with MgSO4. Keep the solution and the brain on ice.</li>
	<li>Following brain removal, under a dissection microscope, dissect out the cerebellum from the brain while still in MgSO4 supplemented dissection solution, and remove the meninges using fine forceps. Turn the cerebellum to its ventral side and insure removal of the choroid plexus.</li>
	<li>Pool the cerebella into a 35-mm dish containing ~1 mL of MgSO4-supplemented dissection solution.</li>
	<li>Chop the tissue into small pieces and transfer it into a 50 mL tube containing 30 mL of MgSO4 buffered dissection solution.</li>
</ol>
3. Mouse Cerebellar Granule Neuron Isolation and Culturing
<ol>
	<li>Centrifuge the 50 mL tube containing the chopped cerebral tissue for 5 min at 644 x g and 4 &#176;C.</li>
	<li>Remove the supernatant and add 10 mL of trypsin dissection solution. Then shake the tube at high speed for 15 min at 37 &#176;C.</li>
	<li>Add 10 mL of trypsin inhibitor solution 1 to the tube and rock gently for 2 min.</li>
	<li>Centrifuge the tube for 5 min at 644 x g and 4 &#176;C.</li>
	<li>Remove the supernatant and add 2 mL of trypsin inhibitor solution 2, and then transfer to a 15 mL tube.</li>
	<li>Triturate the tissue in the 15 mL tube until the solution becomes murky. Then let settle for 5 min.</li>
	<li>Remove the clear supernatant and transfer the supernatant to a new tube containing 1 mL of CaCl2 supplemented dissection solution.</li>
	<li>Add another mL of trypsin inhibitor solution 2 to the bottom of the tube containing the pellet from step 3.6. Triturate again and let settle for 5 min. Remove the supernatant and add it to the tube containing the supernatant from step 3.7 (That is a repeat of steps 3.6-3.7). Repeat this process until most of the tissue is mechanically dissociated.</li>
	<li>Add 0.3 mL of CaCl2-supplemented dissection solution to the supernatant collection for every mL of supernatant.</li>
	<li>Mix the tube contents and then centrifuge for 5 min at 644 x g.</li>
	<li>Remove the supernatant and add 10 mL of fresh media to the pellet and mix.</li>
	<li>Cells may then be counted and diluted to a concentration of 1.5 x 106 cells/mL. Please note that in general, 10 million cells are expected from each dissected brain, dependent on trypsinization time and efficiency of dissection. Plate cells on previously made poly D-lysine plates. For 4-well plates, plate 0.5 mL, giving 7.5 x 105 cells per well. For 35-mm dishes, plate 4 mL, giving 6 x 106 cells per plate.</li>
	<li>After 24 h, add cytosine-&#946;-arabino furanoside (AraC) to the plates to reduce glial contamination. For each mL of media 0.5 &#181;L of 20 mM AraC is required. Addition of AraC does not require a media change. If cells are to be maintained for 7-8 days, repeat this treatment on day 3.</li>
	<li>Maintain cultures in a 5% CO2 incubator at 37 &#176;C and feed with 100 &#181;L of 100 mM glucose added to the culture for every 2 mL of media every 2 days past 5 days. A complete media change is not required.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Distilled water</td>
			<td>Gibco</td>
			<td>#15230162</td>
			<td></td>
		</tr>
		<tr>
			<td>200 mM L-Glutamine</td>
			<td>Gibco</td>
			<td>#25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm Nunc culture dishes</td>
			<td>Gibco</td>
			<td>#174913</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA V Solution</td>
			<td>Sigma Aldrich</td>
			<td>#A-8412</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2 &#8226; 2H2O</td>
			<td>Sigma Aldrich</td>
			<td>#C-7902</td>
			<td></td>
		</tr>
		<tr>
			<td>Chicken Egg White Trypsin Inhibitor</td>
			<td>Sigma Aldrich</td>
			<td>#10109878001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytosine beta-D-Arabino Furanoside</td>
			<td>Sigma Aldrich</td>
			<td>#C-1768</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma Aldrich</td>
			<td>#G-7528</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase1</td>
			<td>Sigma Aldrich</td>
			<td>#11284932001</td>
			<td></td>
		</tr>
		<tr>
			<td>Eagle-minimal essential medium</td>
			<td>Sigma Aldrich</td>
			<td>#M-2279</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma Aldrich</td>
			<td>#G-5417</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat inactivated dialyzed Fetal Bovine Serum</td>
			<td>Sigma Aldrich</td>
			<td>#F-0392</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes Buffer</td>
			<td>Sigma Aldrich</td>
			<td>#H-0887</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide</td>
			<td>Sigma Aldrich</td>
			<td>#216763</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mg/mL Gentamycin</td>
			<td>Sigma Aldrich</td>
			<td>#G-1397</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma Aldrich</td>
			<td>#M-2643</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Methyl-D-aspartic acid</td>
			<td>Sigma Aldrich</td>
			<td>#M-3262</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol Red Solution</td>
			<td>Sigma Aldrich</td>
			<td>#P-0290</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma Aldrich</td>
			<td>#T-4549</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced Serum Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>31985070</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>VWR</td>
			<td>#CABDH9258</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>VWR</td>
			<td>#CABDH9286</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4H2O</td>
			<td>VWR</td>
			<td>#CABDH9298</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly D-lysine</td>
			<td>VWR</td>
			<td>#89134-858</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Wisent</td>
			<td>#319-005-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Wisent</td>
			<td>#080-450</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and culture of mouse primary cerebellar granule neurons

Source: Laaper, M., et al. <a href="https://app.jove.com/v/55871">Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons.</a> J. Vis. Exp. (2017)
This video demonstrates the technique for isolating and culturing primary mouse cerebellar granule neurons. Cells are treated with an antimetabolite to eliminate proliferating glial cells and produce a pure culture of cerebellar granule neurons.

<img alt="Figure 1" src="/files/ftp_upload/22366/22366_Figure1.jpg" /> 
Figure 1: Removal of mouse brain and dissection of cerebellum. (A) To extract the brain of a 6-7 day old mouse, using a pair of forceps, grasp the head and cut the skin anteriorly along the dotted lines using a pair of microdissection scissors. Be careful to cut only the skin and connective tissue, too deep an incision may puncture the skull and damage the brain. These three inci...

Isolation and Culture of Mouse Primary Cerebellar Granule Neurons

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Experimental Preparation
NOTE: ...

Take a freshly harvested mouse pup brain.
Separate the cerebellum and remove its membranous layer.
From the ventral side, remove the network of blood vessels.
Chop the cerebellum into fragments and transfer them to a tube containing buffer.
Centrifuge and discard the supernatant containing debris.
Add trypsin, a proteolytic enzyme, to digest the tissue&#39;s extracellular matrix and loosen the cells.
Add a buffer containing trypsin inhibitor and DNase.
The inhibitor stops trypsin activity while DNase degrades any contaminating DNA.
Mechanically dissociate the tissue to form a cell suspension, including, cerebellar granule neurons and non-neuronal or glial cells.
Transfer the cell suspension to a tube.
Add buffer. Centrifuge and remove the supernatant. Resuspend cells in media and plate them onto culture plates coated with poly-D-lysine.
The cells attach to the coated surfaces.
Add an antimetabolite to eliminate proliferating glial cells and generate a pure culture of cerebellar granule neurons.

isolation-and-culture-of-mouse-primary-cerebellar-granule-neurons

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Primary Neuronal Culture

161 次观看. 来源:Laaper， M. 等人 小鼠原代小脑颗粒神经元神经元死亡和变性建模。J. Vis. Exp. （2017 年） 该视频演示了分离和培养原代小鼠小脑颗粒神经元的技术。用抗代谢物处理细胞以消除增殖的神经胶质细胞并产生小脑颗粒神经元的纯培养物。 

视频: 小鼠原代小脑颗粒神经元的分离和培养 - 概念

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

第一次混合胶质细胞培养的成年小鼠脊髓组织准备

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

科研

JoVE Journal

神经科学

Isolation and Expansion of Neurospheres from Postnatal (P1&#8722;3) Mouse Neurogenic Niches

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including proliferation, self-renewal and multipotency. In the postnatal and adult brain, NSPCs are mainly present in two neurogenic niches: the subventricular zone (SVZ) lining the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus (DG). The isolation of the neurogenic niches from postnatal brain allows obtaining a higher amount of NSPCs in culture with a consequent advantage of higher yields. The close contact between cells within each neurosphere creates a microenvironment that may resemble neurogenic niches. Here, we describe, in detail, how to generate SVZ- and DG-derived neurosphere cultures from 1&#8722;3-day-old (P1&#8722;3) mice, as well as passaging, for neurosphere expansion. This is an advantageous approach since the neurosphere assay allows a fast generation of NSPC clones (6&#8722;12 days) and contributes to a significant reduction in the number of animal usage. By plating neurospheres in differentiative conditions, we can obtain a pseudomonolayer of cells composed of NSPCs and differentiated cells of different neural lineages (neurons, astrocytes and oligodendrocytes) allowing the study of the actions of intrinsic or extrinsic factors on NSPC proliferation, differentiation, cell survival and neuritogenesis.

In this article, we describe, in detail, a protocol for the generation of neurosphere cultures from postnatal mouse neural stem cells derived from the main mouse neurogenic niches. Neurospheres are used to identify neural stem cells from brain tissue allowing the estimation of precursor cell numbers. Moreover, these 3D structures can be plated in differentiative conditions, giving rise to neurons, oligodendrocytes and astrocytes, allowing the study of cell fate.

产后神经球的分离和扩张 （P1+3） 小鼠神经原性球

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including ...

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the cytoplasm or the release of intracellular Ca2+ stores. Pirt-GCaMP3-based Ca2+ imaging of primary sensory neurons of the dorsal root ganglion (DRG) in mice offers the advantage of simultaneous measurement of a large number of cells. Up to 1,800 neurons can be monitored, allowing neuronal networks and somatosensory processes to be studied as an ensemble in their normal physiological context at a populational level in vivo. The large number of neurons monitored allows the detection of activity patterns that would be challenging to detect using other methods. Stimuli can be applied to the mouse hindpaw, allowing the direct effects of stimuli on the DRG neuron ensemble to be studied. The number of neurons producing Ca2+ transients as well as the amplitude of Ca2+ transients indicates sensitivity to specific sensory modalities. The diameter of neurons provides evidence of activated fiber types (non-noxious mechano vs. noxious pain fibers, A&#946;, A&#948;, and C fibers). Neurons expressing specific receptors can be genetically labeled with td-Tomato and specific Cre recombinases together with Pirt-GCaMP. Therefore, Pirt-GCaMP3 Ca2+ imaging of DRG provides a powerful tool and model for the analysis of specific sensory modalities and neuron subtypes acting as an ensemble at the populational level to study pain, itch, touch, and other somatosensory signals.

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

This protocol describes the surgical exposure of the dorsal root ganglion (DRG) followed by GCaMP3 (genetically-encoded Ca2+ indicator; Green Fluorescent Protein-Calmodulin-M13 Protein 3) Ca2+ imaging of the neuronal ensembles using Pirt-GCaMP3 mice while applying a variety of stimuli to the ipsilateral hind paw.

体内 完整背根神经节中初级感觉神经元网络中神经元集合体的钙成像

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the ...

Isolation and Culture of Mouse Primary Cerebellar Granule Neurons

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Isolation and Culture of Mouse Primary Cerebellar Granule Neurons