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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Extracellular Matrix Gel Spots
<ol>
	<li>Perform the following steps one day before the isolation:
	<ol>
		<li>Thaw an aliquot of extracellular matrix gel (100 &#956;l) at 4 &#176;C for at least 1.5 hr. Dilute 1:10 in Dulbecco&#39;s modified Eagle&#39;s medium; with 4,500 mg/L glucose, 4 mM L-glutamine, and 110 mg/ml sodium pyruvate (DMEM). Keep the extracellular matrix gel at 4 &#176;C at all times. 
		NOTE: Abrupt temperature changes will result in uneven coating and crystal formation.</li>
		<li>Keep the diluted extracellular matrix gel solution on ice for 15 min.</li>
		<li>Pre-chill a 20 &#956;l micropipette for 10 min.</li>
		<li>Put 8-well chamber slides into a 100 mm Petri dish and transfer the dish onto a cold surface (e.g. a freezer pack) for 10 min.</li>
		<li>Use the pre-chilled micropipette to put a drop of 10 &#956;l extracellular matrix gel in each well. Keep the Petri dish on the cold surface for at least another 7 min (Figure 1A).</li>
		<li>Completely remove the remaining extracellular matrix gel (Figure 1B) and dry the wells at 37 &#176;C overnight.</li>
	</ol>
	</li>
</ol>
2. Dissection of Head Muscles (Masseter, Digastric, and Levator Veli Palatini)
<ol>
	<li>Before dissection, prepare 50 ml of phosphate-buffered saline (PBS) supplemented with 2% Penicillin-Streptomycin (P/S). Keep on ice.</li>
	<li>After the euthanasia of one young adult rat (9 weeks) with CO2/O2, decapitate the head and remove the skin from the head. Transfer the head to ice-cold PBS supplemented with 2% P/S in a 50 ml tube.</li>
	<li>Masseter muscle (derived from the 1st branchial arch)
	<ol>
		<li>Place the head with one side up on a silicone pad and fix it with hypodermic needles (Figure 2A).</li>
		<li>Identify the parotid gland and the facial nerve (Figure 2A). Expose the deep fascia covering the gland. Cut the fascia and remove the gland using dissection scissors. Identify the external auditory canal. Trace the facial nerve from the stylomastoid foramen and carefully remove the temporal, zygomatic, and buccal branches with a scalpel blade No. 15.</li>
		<li>Free the superficial head of the masseter muscle by removing the fascia. Identify both superficial and deep heads of the masseter muscle. Trace the superficial head until its thick tendinous aponeurosis is inserted in the zygomatic process of the maxilla.</li>
		<li>Separate the tendon from its origin at the zygomatic process with straight forceps. Cut it with a scalpel blade No. 15 or dissection scissors and carefully lift it (Figure 2B).</li>
		<li>Dissect the superficial head of the masseter until its insertion at the angle and inferior half of the lateral surface of the ramus of the mandible with a scalpel blade No. 15 (Figure 2C). Now, completely remove the muscle.</li>
	</ol>
	</li>
	<li>Posterior belly of the digastric muscle (derived from the 2nd branchial arch)
	<ol>
		<li>Place the head in a supine position on the silicone pad and fix it with hypodermic needles (Figure 3A).</li>
		<li>Remove the subcutaneous fat overlying both sublingual and submandibular glands. Next, remove the superficial fascia and glands using dissection scissors. Expose the digastric muscle (anterior and posterior belly).</li>
		<li>Hold the anterior tendon of the posterior belly with straight forceps, cut it, and dissect it carefully until its origin is in the tympanic bulla (Figure 3B). Do the same on the contralateral side.</li>
	</ol>
	</li>
	<li>Levator veli palatini muscle (derived from the 4th branchial arch)
	<ol>
		<li>After dissection of the posterior belly of the digastric muscle, localize the stylohyoid muscle, pull it laterally, and carefully remove it (Figure 4A).</li>
		<li>Localize the tendon of the levator veli palatini that inserts at the tympanic bulla (Figure 4A). Dissect it carefully and cut it on both sides.</li>
		<li>Look for the trachea and the esophagus that runs behind it. Lift the esophagus and expose the pharynx and the larynx.</li>
		<li>Localize and dissect the area of the superior pharyngeal constrictor muscle. Identify the levator veli palatini and cut it at both sides (Figure 4B). 
		NOTE: Directly after dissection, carefully remove tendon and connective tissue from each muscle under the stereo microscope. Submerge all specimens quickly in 70% ethanol and transfer them to ice-cold PBS supplemented with 2% P/S in a 15 ml tube.</li>
	</ol>
	</li>
</ol>
3. Isolation of Satellite Cells (SCs)
<ol>
	<li>Perform the following preparation steps for SC isolation from the three muscle groups:
	<ol>
		<li>Prepare 7.5 ml of 0.1% pronase in DMEM. Filter the solution through a 0.22 &#956;m filter. Pre-warm the solution at 37 &#176;C in a water bath for 10 min before isolation.</li>
		<li>Prepare 35 ml of DMEM supplemented with 10% Horse Serum (HS) and 1% P/S. Also, pre-warm at 37 &#176;C in a water bath.</li>
		<li>Prepare 15 ml culture medium which consists of DMEM supplemented with 20% fetal bovine serum (FBS), 10% HS, 1% P/S, and 1% chicken embryo extract (CEE). Pre-warm at 37 &#176;C in a water bath.</li>
		<li>Pre-coat six plastic pipettes (10 ml) with HS and dry for at least 10 min before use.</li>
	</ol>
	</li>
	<li>In the culture hood, transfer each muscle into a well of a 6-well plate. Using the dissection scissors, cut the muscle in small pieces of about 2 mm. Be careful not to mince the tissue too much.</li>
	<li>Carefully add 2.5 ml of 0.1% pronase solution to each well and incubate at 37 &#176;C for 60 min. Gently shake the plate after 20, 40, and 60 min. 
	NOTE: The exact duration of the incubation depends on factors like the age and strain of the animals.</li>
	<li>Monitor under the microscope. Check the muscle fragments and stop the enzymatic digestion when the fiber bundles appear loosened (Figure 5).</li>
	<li>Add 2.5 ml of DMEM supplemented with 10% HS and 1% P/S. Transfer to a 15 ml tube and centrifuge the tubes at 400 x g for 5 min. Discard the supernatant by decantation.</li>
	<li>Add 5 ml DMEM supplemented with 10% HS and 1% P/S. Pipette the solution up and down with a 10 ml plastic pipette (trituration) at least 20 times to homogenize the tissue.</li>
	<li>Centrifuge the tubes at 200 x g for 4 min. Collect the supernatant and transfer it into a 15 ml tube.</li>
	<li>Add 5 ml DMEM supplemented with 10% HS and 1% P/S. Pipette again with a 10 ml plastic pipette until the tissue fragments pass easily.</li>
	<li>Centrifuge the tubes at 200 x g for 4 min and collect the supernatant in a 15 ml tube.</li>
	<li>Put a cell strainer (40 &#956;m) onto a 50 ml tube and transfer the supernatant containing the dissociated cells onto the filter. Wash with 1 ml DMEM for maximal cell recovery.</li>
	<li>Centrifuge the tubes at 1,000 x g for 10 min and discard the supernatant with a pipet.</li>
	<li>Resuspend the pellet in 300 &#956;l culture medium and count the cells in a hemocytometer.</li>
</ol>
4. Differentiation of Satellite Cells on Extracellular Matrix Gel Spots
<ol>
	<li>Dilute the cell suspension to obtain 1.5 x 103 cells in 10 &#956;l of culture medium.</li>
	<li>Secure the covers of the chamber slides with tape and mark the spots with a black marker on the bottom side of the object glass.</li>
	<li>Using a micropipette, put a drop of 10 &#956;l cell suspension onto the extracellular matrix gel spot. Check under the microscope whether the drop of cell suspension has been placed correctly on the spot. Incubate for six hours at 37 &#176;C.</li>
	<li>Carefully add 400 &#956;l of culture medium (DMEM supplemented with 20% FBS, 10% HS, 1% P/S, and 1% CEE) and incubate for three days at 37 &#176;C. 
	NOTE: At this point, freshly isolated SC are subjected to massive trauma (enzymatic digest and harsh trituration) and they need to recover. Do not disturb the cells during the first three days. Next, the culture medium can be changed depending on the type of experiment. 
	The extracellular matrix gel spots can be seeded with a high cell density (1.5-2.5 x 103/20 &#956;l) for differentiation assay. Culture medium (DMEM supplemented with 20% FBS, 10% HS, 1% P/S, and 1% chicken embryo extract) can be replaced every third day.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Hypodermic Needle 25 G 
			&#160;0.5 x 25 m</td>
			<td>BD Microlance</td>
			<td>300400</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scissors</td>
			<td>Braun</td>
			<td>BC154R</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro forceps straight</td>
			<td>Braun</td>
			<td>BD330R</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scalpel Blade No. 15</td>
			<td>Swann-Morton</td>
			<td>0205</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol 70%</td>
			<td>Denteck</td>
			<td>2,010,005</td>
			<td></td>
		</tr>
		<tr>
			<td>Permanox Slide, 8 Chamber</td>
			<td>Thermo Scientific</td>
			<td>177445</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well cell culture plate</td>
			<td>Greiner bio-one</td>
			<td>657160</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Dishes (100 x 20 mm)</td>
			<td>Greiner bio-one</td>
			<td>664160</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml sterile conical centrifuge tube</td>
			<td>BD Biosciences</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml sterile conical centrifuge tube</td>
			<td>BD Biosciences</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer (40 &#956;m)</td>
			<td>Gibco</td>
			<td>431750</td>
			<td></td>
		</tr>
		<tr>
			<td>10 ml serological pipette</td>
			<td>Greiner bio-one</td>
			<td>607180</td>
			<td></td>
		</tr>
		<tr>
			<td>20 &#181;l FT20</td>
			<td>Greiner bio-one</td>
			<td>774288</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel, Phenol-Red Free</td>
			<td>BD Biosciences</td>
			<td>356237</td>
			<td>10 ml</td>
		</tr>
		<tr>
			<td>Pronase</td>
			<td>Calbiochem</td>
			<td>53702</td>
			<td>10 KU</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium, high glucose, GlutaMAX Supplement, pyruvate</td>
			<td>Gibco</td>
			<td>10569-010</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Fisher Scientific</td>
			<td>3600511</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>Gibco</td>
			<td>26050088</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/ ml)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>100 ml</td>
		</tr>
		<tr>
			<td>Chicken Embryo Extract</td>
			<td>MP Biomedicals</td>
			<td>2850145</td>
			<td>20 ml</td>
		</tr>
	</tbody>
</table>

a technique to isolate satellite cells from rat head muscle tissue

Source: Carvajal Monroy, P. L., et al. <a href="https://app.jove.com/v/52802">Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles.</a> J. Vis. Exp. (2015).
This video demonstrates the isolation of satellite cells from rat head branchiomeric muscle tissues through tissue dissection, enzymatic digestion, and subsequent mechanical and centrifugal purification. The cells are then seeded on extracellular matrix-coated wells for differentiation into myoblasts, and eventually into myotubes and mature muscle fibers.

<img alt="Figure 1" src="/files/ftp_upload/22530/22530_Figure1.jpg" /> 
Figure 1: Extracellular matrix gel spots in a chamber slide. (A) For easy manipulation, place the 8-well chamber slide into a 100 mm Petri dish. Pipet 10 &#956;l extracellular matrix gel in each chamber and put it on a cold surface (7 min). (B) Chamber slide after the excess extracellular matrix gel is removed.
<img alt="Figure 2" src="/files/ftp_upload/22530/22530_Figure2.jpg" /> 
Figure 2: Dissection of the masseter muscle. (A) Head of the animal in a lateral view. Ear (E), Parotid gland (P), and facial nerve (VII). (B) Tendinous aponeurosis (Te) of the superficial head of the masseter muscle (Ms) and temporal muscle (T). Separate the tendon from its insertion with forceps. (C) Carefully dissect the muscle until its insertion at the ramus of the mandible. E: ear, P: parotid gland, VII: facial nerve, T: Temporal muscle, Ms: superficial head of the masseter muscle, Te: tendon, Mp: deep head of the masseter muscle.
<img alt="Figure 3" src="/files/ftp_upload/22530/22530_Figure3.jpg" /> 
Figure 3: Dissection of the posterior belly of the digastric muscle. (A) Head of the animal in a supine position. Localize the submandibular gland (Sg), masseter muscle (M), facial nerve (VII), and sternocleidomastoid muscle (SCM). Remove the submandibular gland. (B) Localize the digastric muscle anterior (AD) and posterior belly (PD). With straight forceps, take the anterior tendon of the posterior belly, cut it, and dissect it carefully until its origin in the tympanic bulla (ty). E: ear, Sg: submandibular gland, VII: facial nerve, M: masseter muscle, SMC: sternocleidomastoid muscle, AD: anterior belly digastric muscle, PD: posterior belly digastric muscle, Ty: Tympanic bulla.
<img alt="Figure 4" src="/files/ftp_upload/22530/22530_Figure4.jpg" /> 
Figure 4: Dissection of the levator veli palatini muscle. (A) General view after dissection of the digastric muscle (posterior belly). Stylohyoid muscle (St) and tendon of the levator veli palatini can be localized. Note the trachea (T) and esophagus (Es) running behind it. (B) After lifting the trachea and the esophagus the pharynx (P) is exposed. The levator veli palatini that runs laterally towards the soft palate is now visible. The arrow indicates the dissected superior pharyngeal constrictor muscle; note the levator veli palatini muscles on both sides. E: ear, St: stylohyoid muscle, VII: facial nerve, M: masseter muscle, AD: anterior belly digastric muscle, PD: posterior belly digastric muscle, T: trachea, Es: esophagus, P: Pharynx, *levator veli palatini muscle.
<img alt="Figure 5" src="/files/ftp_upload/22530/22530_Figure5.jpg" /> 
Figure 5: Appearance of the muscle tissue (A) before and (B) after enzymatic digestion with pronase. Note that muscle bundles appear to be loosened after enzymatic digestion.

A Technique to Isolate Satellite Cells From Rat Head Muscle Tissue

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Extracellular Matrix Gel Spots

	...

Begin with a rat head muscle and chop it into fragments.
Add a cocktail of proteolytic enzymes and incubate with gentle shaking.
The enzymes digest the tissue&#39;s extracellular matrix, loosening the muscle fibers.
Add media containing serum proteins to inactivate the enzymes.
Transfer the tissue to a tube, centrifuge, and discard the supernatant.
Resuspend the tissue in media. Repeat mechanical disruption and centrifugation twice to obtain a homogeneous suspension of satellite cells &#8212; stem cells crucial for muscle repair and regeneration.
Collect the satellite cell-containing supernatant.
Pass through a cell strainer to obtain a single-cell suspension. Centrifuge, discard the supernatant, and resuspend the cells in media.
Place drops of the cell suspension onto extracellular matrix protein-coated chamber slide wells.
The coated surface facilitates satellite cell attachment. Add media to the wells.
Growth factors in the media induce satellite cell proliferation and differentiation into myoblasts&#160; &#8212; muscle cell precursors, which further proliferate and fuse to form multinucleated myotubes.
The myotubes eventually mature into muscle fibers.

a-technique-to-isolate-satellite-cells-from-rat-head-muscle-tissue

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

58 次观看. 来源:Carvajal Monroy， P. L.， et al.大鼠头部鳃肌卫星细胞的分离和表征。J. Vis. Exp.（2015 年）。 该视频演示了通过组织解剖、酶消化以及随后的机械和离心纯化从大鼠头部鳃肌组织中分离卫星细胞。然后将细胞接种在细胞外基质包被的孔上，以分化为成肌细胞，并最终分化为肌管和成熟肌纤维。 

视频: 一种从大鼠头部肌肉组织中分离卫星细胞的技术 - 概念

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

从人类骨骼肌成肌原细胞和成纤维细胞的分离和定量免疫细胞化学表征

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

科研

JoVE Journal

发育生物学

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

使用分离线粒体从小鼠骨骼肌进行高通量微孔板呼吸测量最小数量

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting occurs. Loss of muscle mass and function is associated with muscle fiber type atrophy, fiber type switching, defective muscle regeneration associated with dysfunction of satellite cells, muscle stem cells, and other pathophysiological processes. These changes are associated with changes in intracellular as well as local and systemic niches. In addition to most commonly used rodent models of muscle ageing, there is a need to study muscle homeostasis and wasting using human models, which due to ethical implications, consist predominantly of in vitro cultures. Despite the wide use of human Myogenic Progenitor Cells (MPCs) and primary myoblasts in myogenesis, there is limited data on using human primary myoblast and myotube cultures to study molecular mechanisms regulating different aspects of age-associated muscle wasting, aiding in the validation of mechanisms of ageing proposed in rodent muscle. The use of human MPCs, primary myoblasts and myotubes isolated from adult and aged people, provides a physiologically relevant model of molecular mechanisms of processes associated with muscle growth, atrophy and regeneration. Here we describe in detail a robust, inexpensive, reproducible and efficient protocol for the isolation and maintenance of human MPCs&#160;and their progeny&#160;&#8212; myoblasts and myotubes from human muscle samples using enzymatic digestion. Furthermore, we have determined the passage number at which primary myoblasts from adult and aged people undergo senescence in an in vitro culture. Finally, we show the ability to transfect these myoblasts and the ability to characterize their proliferative and differentiation capacity and propose their suitability for performing functional studies of molecular mechanisms of myogenesis and muscle wasting in vitro.

This protocol describes a robust, reproducible and simple method of isolation and culture of myoblast progenitor cells from the skeletal muscle of adult and aged people. The muscles used here include foot and leg muscles. This approach enables the isolation of an enriched population of primary myoblasts for functional studies.

制备及文化活动从成年和老年人类骨骼肌成肌前体细胞/成肌细胞主要的

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting ...

A Technique to Isolate Satellite Cells From Rat Head Muscle Tissue

成績單

A Technique to Isolate Satellite Cells From Rat Head Muscle Tissue