The authors would like to extend many thanks to Drs. Josep Planas and Juan Castillo for their professional expertise in the development and application of this culture protocol to small fishes and amphibians. Thanks are also due to the countless individuals who have tirelessly assisted with the dissection and dissociation of muscle tissue from many fish (both in species and number), including Matthew Charging, Delci Christensen, Zachary Fowler, Brooke Franzen, Nathan Froehlich, Kira Marshall, Ben Meyer, Ethan Remily, and Sinibaldo Romero. This work was supported by University of Alabama at Birmingham Department of Biology start-up funds, Center for Protease Research NIH Grant # 2P20 RR015566, NIH NIAMS Grant # R03AR055350, and NDSU Advance FORWARD NSF Grant #HRD-0811239 to PRB. Support was also provided by the UAB Nutrition Obesity Research Center award # P30DK056336, NIH NIDDK. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

Ethics Statement: All experimentation involving vertebrate animals described herein was approved in advance by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham and is consistent with guidelines established by the Office of Laboratory Animal Welfare, National Institutes of Health of the U.S. Department of Health and Human Services.
1. Preparation for Culture
<ol>
	<li>Prepare the base medium as follows: 9 mM NaHCO3 (1.51 g per 2 L), 20 mM HEPES (9.53 g per 2 L) in DMEM (13.48 g/L; 26.96 g per 2 L).
	<ol>
		<li>Determine pH and adjust to 7.40 with HCl or NaOH.</li>
		<li>Determine osmolality and adjust to ~ 300 mOsm with NaCl (if needed).</li>
		<li>Filter sterilize using (2) 1 L vacuum sterilization systems and store at 4 &#176;C protected from light for up to 2-3 months. 
		NOTE: See Tables 1 and 2 for complete reagent, tools, consumables, and equipment information.</li>
	</ol>
	</li>
	<li>Prepare poly-L-lysine-treated plates by dissolving 5 mg of &#947;-irradiated poly-L-lysine (&#62; 300,000 mw) in 50 ml&#160;of ultrapure sterile water. Mix gently by inversion for 10 min at room temperature, ensuring full dissolution of lysine polymers.</li>
	<li>Pipette the necessary amount of poly-L-lysine solution into each well, according to the plate type (Table 3). Allow plates to stand in laminar flow hood for 5 minutes.</li>
	<li>Next, remove poly-L-lysine solution and wash twice with sterile water. Allow plates to air dry in laminar flow hood for 20 minutes.</li>
	<li>Obtain 1 mg of laminin (Engelbreth-Holm-Swarm origin) and dissolve in 50 ml&#160;of base medium to a final concentration of 20 &#956;g/ml. Apply laminin solution to poly-L-lysine-treated cell culture plates at 2 &#956;g/cm2. (see Table 3 for different plate sizes)</li>
	<li>Gently swirl the solution to ensure full well coverage. Seal plates with laboratory tape and place in incubator for 24 hours. The incubator should be set to appropriate temperature for species being used (see Table 7), and no additional gases need to be added to incubator. 
	NOTE: Laminin must be allowed to adhere to plates for 24 hours before seeding cells. Failure to do so will result in poor to no cell adherence.</li>
	<li>Prepare media as described in Table 4. Complete media is best prepared the day of use.</li>
	<li>In preparation for tissue isolation, assemble the following items and autoclave: 300-500 ml&#160;beaker(s); glass Petri dishes; scalpel handles; forceps (fine and coarse); dissection scissors; and several sheets of water-repellant autoclave paper. 
	NOTE: For each person assisting with the dissection process, (2) scalpel handles, (1) forceps (type depending on personal preference), (1) dissection scissors, and (5-10) sheets of water-repellant autoclave paper are sufficient.</li>
	<li>In addition to the autoclaved items above, assemble 70% ethanol, a weight balance, sterile 50 ml&#160;conical tubes (number depends on size of culture), and ice.</li>
</ol>
2. Tissue Dissection
<ol>
	<li>Before beginning, ensure that a sufficient stock of fish is available. 
	NOTE: The age of the fish to be used is of critical importance. While fish of two different species may be of similar weights, a younger fish of one species will possess more MPCs than an older fish of another species. As a general rule, younger fish, especially when working with salmonids, are best. For danionins, fish as old as one year can be utilized optimally, while salmonids of age 4-6 months or younger (up to 15 g) are optimal.</li>
	<li>For each 5 g of tissue to be dissected, aliquot 25 ml&#160;of isolation medium into a sterile 50 ml&#160;conical tube.</li>
	<li>Weigh, record mass, and number the tube(s). Place tubes on ice.</li>
	<li>Euthanize 2-3 fish at a time by immersion in &#62; 300 mg/L sodium bicarbonate-buffered tricaine methanesulfonate. Allow opercular movement to cease for &#62; 5 minutes and then submerse fish in 70% ethanol (contained in the sterile beakers) for 30 seconds.</li>
	<li>Remove fish from 70% ethanol and place on water-repellant autoclave paper. Directly behind the opercula, use a scalpel to make a superficial, shallow incision. Remove the scales and skin by grasping the skin at the aforementioned incision and pull toward tail of fish.</li>
	<li>Following skin removal, excise the epaxial, fast-glycolytic &#8216;white&#8217; muscle of the fish from both sides (avoiding the slow-oxidative &#8216;red&#8217; muscle located near the lateral line) and place in isolation medium. Discard the remainder of the fish as required by institution. 
	NOTE: While it is entirely possible to culture MPCs originating from red muscle, nearly every publication using teleost MPCs has utilized cells isolated from the epaxial myotome.</li>
	<li>Repeat steps 2.4-2.6 until a sufficient quantity of muscle is obtained. During dissection, ensure that pooled muscle tissue remains on ice to maintain cell viability.</li>
</ol>
3. Mechanical Dissociation
<ol>
	<li>Pour one tube of 25 ml/5 g of muscle tissue into a glass Petri dish. Using two scalpel handles with attached #10 scalpel blades, mince tissue to a slurry or pur&#233;e consistency. A &#8220;back-and-forth&#8221; motion of pulling the scalpel blades past each other works best. 
	NOTE: While tissue homogenizers may seem appropriate, the number of viable cells will be dramatically reduced, if not abolished.</li>
	<li>Using a 25 ml&#160;serological pipette and serological pipettor, remove the slurried tissue and replace in the original conical tube.</li>
	<li>Repeat steps 3.1-3.2 until all tissue in all tubes has been mechanically dissociated.</li>
	<li>Centrifuge tissue for 5 minutes at 300 x&#160;g and 10 &#176;C.</li>
	<li>Following centrifugation, discard the 25 ml&#160;of media supernatant with a 25 ml&#160;serological pipette, vacuum aspirator, or by careful decanting.</li>
	<li>Add 25 ml&#160;of wash medium to each tube, resuspend tissue, and recentrifuge as above.</li>
	<li>Wash twice with wash medium, removing the supernatant each time.</li>
</ol>
4. Enzymatic Dissociation
<ol>
	<li>During step 3.4, prepare collagenase solution by combining 0.22 g of Type IV collagenase and 44 ml&#160;of base medium (or sufficient quantity at 0.11 g/0.22 ml).</li>
	<li>Stir in a beaker for 10-15 minutes at 4 &#176;C. Filter sterilize with a 50 ml&#160;syringe and a 0.45 &#956;m syringe filter. 
	NOTE: More than one syringe filter may be needed, depending on the collagenase preparation. Collagenase obtained from vendors other than Worthington Biochemical poses more difficulty during filtration. Collagenase is used to dissociate peptide bonds in collagen that constitute the endomysium. This digestion will remove that protective barrier of the myofibers.</li>
	<li>For each g of muscle tissue, resuspend tissue in a 0.2% collagenase solution. For 5 g of muscle tissue, add 10 ml&#160;of collagenase solution and 15 ml&#160;of base medium.</li>
	<li>Add 10 &#956;l&#160;of PSF per ml&#160;of collagenase solution/base medium (e.g.&#160;250 &#956;l&#160;to 25 ml). Ensure that tissue is well suspended, as this will affect the efficiency of the enzymatic digestion.</li>
	<li>Incubate muscle tissue in collagenase for 60 minutes at 18 &#176;C with gentle rocking. Depending on the degree of mechanical dissociation and species, collagenase digestion may be extended to 90 minutes, although this may affect cell viability.</li>
	<li>Following collagenase digestion, centrifuge conical tubes at 300&#160;x&#160;g for 5 minutes at 12 &#176;C. Discard the supernatant and resuspend tissue in 25 ml&#160;of wash medium.</li>
	<li>Recentrifuge at 300&#160;x&#160;g for 5 minutes at 12 &#176;C. Repeat with washing medium a second time.</li>
	<li>After washing tissue twice, resuspend muscle tissue in 25 ml&#160;of wash medium and triturate with a 10 ml&#160;serological pipette until the tissue/medium homogenate passes in and out of the pipette with relative ease. 5-10 triturations is usually sufficient.
	<ol>
		<li>Repeat with a 5 ml&#160;serological pipette and then a 16 gauge metal cannula attached to a syringe.</li>
	</ol>
	</li>
	<li>Centrifuge conical tubes at 300&#160;x&#160;g for 5 minutes at 12 &#176;C. Decant supernatant.</li>
	<li>During the above 5 minute centrifugation, prepare the trypsin digestion solution by combining 0.25 g of trypsin with 5 ml&#160;of base medium.</li>
	<li>Stir at 4 &#176;C for 10-15 minutes and filter sterilize with a 20 ml&#160;syringe and 0.45 &#956;m syringe filter.</li>
	<li>Retrieve conical tubes and add 100 &#956;l&#160;of trypsin solution and 4.9 ml&#160;of dissociation medium for every 1 g of muscle tissue to every tube. For example, add 500 &#956;l&#160;of trypsin solution and 24.5 ml&#160;of dissociation medium to 5 g of muscle tissue. 
	NOTE: Trypsin is a serine protease that will separate myogenic precursor cells from the basal lamina.</li>
	<li>Resuspend tissue in the trypsin/dissociation medium well. Incubate for 20 minutes at 18 &#176;C.</li>
	<li>Following the first trypsin incubation, centrifuge conical tubes at 300&#160;x&#160;g&#160;for 1 min at 12 &#176;C.</li>
	<li>Neutralize trypsin by combining the supernatants with isolation medium at a concentration of 1 volume of supernatant to 4 volumes of isolation medium. Store at 4 &#176;C during the second trypsin digestion.</li>
	<li>To the remaining pellet of tissue, repeat steps 4.12-4.15, combining the supernatant following step 4.15 with the supernatant/isolation medium from the first trypsin digestion.</li>
	<li>ALTERNATIVELY: The collagenase and trypsin digestions may be combined to maximize cell yields.
	<ol>
		<li>To do this, triturate the collagenase digest using a metal cannula (6 in length and 16 gauge works best) attached to a 20 ml&#160;syringe until the homogenate easily passes through the cannula.</li>
		<li>To the homogenate, add 500 &#956;l&#160;of trypsin solution (prepared in step 4.10) and incubate at 18 &#176;C for 20 minutes.</li>
		<li>Following the incubation, centrifuge conical tubes at 300 x&#160;g for 1 minute at 12 &#176;C.</li>
		<li>Neutralize trypsin by combining the supernatants with isolation medium at a concentration of 1 volume of supernatant to 4 volumes of isolation medium.</li>
		<li>Store at 4 &#176;C during the second trypsin digestion.</li>
		<li>Proceed with step 4.18 as with the standard protocol.</li>
	</ol>
	</li>
	<li>Dispense the final supernatant/isolation medium mixture into 50 ml conical tubes and centrifuge at 300&#160;x&#160;g for 10 minutes at 12 &#176;C.</li>
	<li>Remove the supernatants taking care not to disturb the cell pellets. Pipette 2 ml&#160;of complete medium&#160;into each tube, dissolving each cell pellet.</li>
	<li>Combine cells suspended in complete medium in one 50 ml&#160;conical tube.</li>
	<li>Rinse each tube with 1 ml&#160;of complete medium, combining with the pool of cells resuspended previously.</li>
	<li>Triturate with a metal cannula and syringe 5-10 times.</li>
	<li>Filter cell suspension through a 100 &#956;m cell strainer, rinsing with complete medium.</li>
	<li>Filter the cell suspension through a 40 &#956;m cell strainer.</li>
	<li>Add sufficient complete medium to 50 ml&#160;and centrifuge at 300&#160;x&#160;g for 10 minutes at 15 &#176;C.</li>
</ol>
5. Counting, Dilution, and Seeding of Cells
<ol>
	<li>Following the final centrifugation in step 4.25, remove supernatant by careful decanting or with a serological pipette. Resuspend the cell pellet in 5 ml&#160;of complete medium.</li>
	<li>With cells fully resuspended, remove 20 &#956;l&#160;of the cell suspension and place in a 1.5 ml&#160;microcentrifuge tube.</li>
	<li>Add 5 &#956;l&#160;of Trypan blue dye and wait 5 minutes. Using a hemocytometer, determine the number of viable cells.</li>
	<li>Upon determination, dilute cells to the needed concentration. Teleost MPCs are best seeded at 150,000-200,000 cells per cm2. For a six-well plating strategy, diluting cells to 1.5 x 106&#160;- 2.0 x 106 per ml&#160;supports sufficient proliferation and differentiation.</li>
	<li>Retrieve poly-L-lysine-treated, laminin-coated plates and seed cells. (See Table 5 for plate-specific dilutions and plating volumes.) NOTE: Table 6 depicts average number of cells isolated per gram of muscle tissue isolated from various teleost species. Rainbow trout (O. mykiss) exhibit fewer MPCs per gram of muscle tissue than do zebrafish (D. rerio) and will thereby require more fish to isolate from for proper seeding.</li>
	<li>Seal plates with laboratory tape and place in chilling incubator at the appropriate temperature. MPCs from all species listed here (save for mice) may be incubated under normal atmospheric conditions without carbon dioxide (CO2) supplementation. (See Table 7.)</li>
	<li>ALTERNATIVELY: Some laboratories have adopted a protocol that calls for allowing MPCs to adhere to the culture substrata for 40 minutes.
	<ol>
		<li>Then rinse plates with washing media to remove any loosely attached and non-adherent cells. Warning: This step is very important to avoid &#8220;non-specific&#8221; adhesion of other cells (e.g.&#160;fibroblasts) to the culture substrata.</li>
		<li>Add complete media and place cells in the incubator and care for as detailed below.</li>
	</ol>
	</li>
</ol>

There is nothing to disclose.

The myogenic program, in whichever species examined, can be most easily studied through an in vitro system. Indeed, upon isolation, myogenic precursor cells (MPCs) in fish or myosatellite cells (MSCs) in mammals readily enter this highly regulated process involving the proliferation, cell cycle withdrawal, and terminal differentiation of myoblasts and the fusion of those myoblasts into nascent myotubes. The general lack of transgenic gene reporter strains of piscine species (with the possible exception of the ze...

Considerable understanding of mammalian myogenesis has been obtained through the recapitulation of this process in both primary mouse (Mus musculus) myoblast cultures and the well-described mouse-derived cell line, C2C125. Beginning in the 1950s6, these cultures have led to much advancement in the understanding of the murinemyogenic program and, by extension, myogenesis in other vertebrates. Additionally, single cell myofiber explant techniques have increased out understanding of interactions between satellite cells and surrounding myofibers7-9.Cell cultures are particularly attractive for investigations of myogenesis due to the short time from precursor to differentiated cell10, relative ease of transfection for RNAi11-14, transgenic15,16 and overexpression studies14,17,18 , in vitro expansion followed by in vivo transplantation18-20, and even comparison of myogenic precursor cells and their regulating agents across taxa21,22. While differences due to the artificial environment of the culture system have been described5,23 , these in vitro systems have proven to be indispensible to our dissection of the intricate program governing the formation of multinucleated, terminally differentiated myofibersfrom mononucleated proliferative progenitor cells known as myosatellite cells (MSCs) among the mammals.
Outside of the class Mammalia, however, the conservation and/or divergence of mechanisms controlling myogenesis are poorly understood, largely due to the difficulty in culturing myogenic precursor cells (MPCs) and myoblasts from various taxa. Indeed, primary myoblast cultures have only been described in three birds24-26, one reptile27, a few amphibians28-30, and some fishes1,3,4,31-33. Continuous myogenic cell lines from vertebrates other than rodents34-36are even more rare, with the only non-mammalian myogenic cell line being derived from Japanese quail (Cortunix japonica), QM737. Despite many attempts at immortalization, a teleost myogenic cell line remains elusive and a protocol for efficient transfection of these cells was only published this year15. Thus, clear and well-optimized protocols for culturing primary MPCs and myoblasts from a variety of vertebrates are very much needed to not only further expand our knowledge of the evolution of the myogenic program, but to employ the power of comparative physiology to make breakthroughs in the treatment of human skeletal muscle diseases and disorders.
While the literature contains many reports of MPC/myoblast isolation38-49, it is common for authors to describe the protocols for such isolations in brief, often incomplete, formats. Further, the most instructive protocols reported have been developed for mice50-53, and some of these rely on antibody selection54,55 or fluorescence transgenes56,57, making these protocols unusable or impractical inmost non-rodent species utilized by muscle biologists. With little known about piscine, amphibian, and reptile myogenesis, a detailed and thorough protocol, described with audiovisual guidance and with demonstrated efficiency in distantly related species, would be most helpful to the field.
First described by Powell and colleagues in 198958, the following protocol was initially developed to isolate MPCs and myoblasts from salmonid fishes (namely, rainbow trout, Oncorhynchus mykiss, and Atlantic salmon, Salmo salar) and some larger cyprinids (i.e.&#160;goldfish, Carassiusauratusauratus). In 2000, Fauconneau and Paboeuf optimized a primary myoblast culture for rainbow trout59, and minoroptimizations made that protocol utilizable in several smaller minnows of the Danioninae clade (zebrafish, Danio rerio, and giant danio, Devario aequipinnatus)32 due to the many genetic tools available for zebrafish work and thus its close relatives. Teleost fish are attractive organisms for study due to their divergent growth strategy (at least in most species). Large salmonids, like most fishes, grow indeterminately, with growth potential unfettered by an asymptote at maturity, even in old age60-62. Unlike zebrafish, large danionins such as the giant danio63 and moustached danio display growth potentials typical of teleost fish, making their direct juxtaposition an ideal platform for understanding whether MPC cell fate choice plays a role in skeletal muscle hyperplasia versus hypertrophy.
Likewise, we have demonstrated that this protocol can be used with mice and axolotls, with relatively high cell yield and viability indices. Urodele salamanders, such as the Mexican axolotl (Ambystomamexicanum),possess the remarkable ability to regenerate tissues, including entire limbs and tails64-66. This characteristic makes these amphibians interesting models of skeletal muscle wasting and aging. Using the protocol described below, a similar approach can be undertaken as has been done in many fish species, providing an even wider comparative context for such studies. As many truly comparative biologists appreciate, the most meaningful advances in basic biology and translational biomedicine can be made when data are analyzed within the widest spectrum (here, the entire vertebrate lineage).

<table border="0" cellpadding="0" cellspacing="0" width="743">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 1. Detailed Reagent Information</td>
		</tr>
		<tr>
			<td height="42" style="height:43px;width:205px;">Reagent</td>
			<td style="width:217px;">Company (Preferred v. Alternate)</td>
			<td style="width:160px;">Catalog Number (Preferred v. Alternate)</td>
			<td style="width:161px;">Quantity per Culture</td>
		</tr>
		<tr>
			<td height="20" style="height:20px;">&#947;-irradiated poly-L-lysine</td>
			<td>Sigma-Aldrich (MP Biomedicals)</td>
			<td>P5899 (ICN19454405)</td>
			<td>5 mg</td>
		</tr>
		<tr>
			<td height="21" style="height:21px;width:205px;">DMEM (high glucose)</td>
			<td>Sigma-Aldrich (cellgro)</td>
			<td>MT-50-003-PB (D7777)</td>
			<td>2 L</td>
		</tr>
		<tr>
			<td height="21" style="height:21px;width:205px;">Laminin</td>
			<td>BD Biosciences (Sigma-Aldrich)</td>
			<td>CB-40232 (L2020)</td>
			<td>1 mg</td>
		</tr>
		<tr>
			<td height="41" style="height:41px;width:205px;">Sodium Bicarbonate (NaHCO3)</td>
			<td>Fisher Scientific (Sigma-Aldrich)</td>
			<td>BP328-500 (S5761)</td>
			<td>1.51 g</td>
		</tr>
		<tr>
			<td height="21" style="height:21px;width:205px;">HEPES (C8H18N2O4S)</td>
			<td>Fisher Scientific (Sigma-Aldrich)</td>
			<td>BP310-1 (H6147)</td>
			<td>9.53 g</td>
		</tr>
		<tr>
			<td height="21" style="height:21px;width:205px;">Antibiotic/Antimycotic</td>
			<td>Thermo Scientific (Sigma-Aldrich)</td>
			<td>SV3007901 (A5955)</td>
			<td>17-20 mL</td>
		</tr>
		<tr>
			<td height="21" style="height:21px;width:205px;">Gentamicin Sulfate</td>
			<td>Lonza (Sigma-Aldrich)</td>
			<td>BW17-519Z (G1397)</td>
			<td>2-3 mL</td>
		</tr>
		<tr>
			<td height="21" style="height:21px;width:205px;">Donor Equine Sera</td>
			<td>Thermo Scientific (Sigma-Aldrich)</td>
			<td>SH3007403 (H1270)</td>
			<td>75 mL</td>
		</tr>
		<tr>
			<td height="21" style="height:21px;width:205px;">Fetal Bovine Sera</td>
			<td>Thermo Scientific (Sigma-Aldrich)</td>
			<td>SH3007103 (F2442)</td>
			<td>25 mL</td>
		</tr>
		<tr>
			<td height="21" style="height:21px;width:205px;">Collagenase (Type IV)</td>
			<td>Worthington (Sigma-Aldrich)</td>
			<td>LS004189 (C9891)</td>
			<td>0.44 g</td>
		</tr>
		<tr>
			<td height="21" style="height:21px;width:205px;">Trypsin (from Pancreas)</td>
			<td>MP Biomedicals (Sigma-Aldrich)</td>
			<td>ICN15357125 (T5266)</td>
			<td>1 g</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 2. Consumables, Tools and Equipment</td>
		</tr>
		<tr>
			<td height="42" style="height:43px;width:239px;">Consumable</td>
			<td style="width:204px;">Tools</td>
			<td style="width:199px;">Equipment</td>
			<td></td>
		</tr>
		<tr>
			<td height="28" style="height:28px;">Cell Culture Plates</td>
			<td>Forceps (Coarse)</td>
			<td>Serological Pipettor</td>
			<td></td>
		</tr>
		<tr>
			<td height="28" style="height:28px;width:239px;">Sterile 50 mL Conical Tubes</td>
			<td style="width:204px;">Forceps (Fine)</td>
			<td>pH Meter</td>
			<td></td>
		</tr>
		<tr>
			<td height="28" style="height:28px;width:239px;">Laboratory Tape</td>
			<td style="width:204px;">Scalpel Handles</td>
			<td>Chilling Incubator (Echotherm)</td>
			<td></td>
		</tr>
		<tr>
			<td height="28" style="height:28px;width:239px;">0.2 &#956;m Vacuum Sterilization Systems</td>
			<td style="width:204px;">Scalpel Blades (#10, #11)</td>
			<td>Laminar Flow Hood</td>
			<td></td>
		</tr>
		<tr>
			<td height="28" style="height:28px;width:239px;">Water-repellant Autoclave Paper</td>
			<td style="width:204px;">Surgical Scissors</td>
			<td>Vacuum Manifold</td>
			<td></td>
		</tr>
		<tr>
			<td height="28" style="height:28px;width:239px;">Serological Pipettes</td>
			<td>Glass Petri Dishes</td>
			<td>Microosmolality Meter</td>
			<td></td>
		</tr>
		<tr>
			<td height="28" style="height:28px;width:239px;"></td>
			<td>12-16 G Cannulas with Luer Locks</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 3. Optimized Volumes for Coating Cell Culture Plates</td>
		</tr>
		<tr>
			<td height="24" style="height:24px;width:167px;">Plate Size</td>
			<td style="width:192px;">cm^2 per Well</td>
			<td style="width:132px;">Poly-L-lysine*</td>
			<td style="width:145px;">Laminin**</td>
		</tr>
		<tr>
			<td height="20" style="height:20px;">6 well</td>
			<td>9.5</td>
			<td>1.6 mL</td>
			<td>1</td>
		</tr>
		<tr>
			<td height="21" style="height:21px;width:167px;">24 well</td>
			<td>1.9</td>
			<td>0.32</td>
			<td>0.2</td>
		</tr>
		<tr>
			<td height="21" style="height:21px;width:167px;">48 well</td>
			<td>0.95</td>
			<td>0.16</td>
			<td>0.1</td>
		</tr>
		<tr>
			<td height="21" style="height:21px;width:167px;">96 well</td>
			<td>0.32</td>
			<td>0.06</td>
			<td>0.03</td>
		</tr>
		<tr>
			<td height="40" style="height:40px;width:167px;">*0.1 mg/mL concentration</td>
			<td colspan="2">** 0.020 mg/mL concentration</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 4. Media for Isolation, Dissociation, and Culture</td>
		</tr>
		<tr>
			<td height="24" style="height:24px;width:163px;">Reagent</td>
			<td style="width:155px;">Isolation</td>
			<td style="width:127px;">Wash</td>
			<td style="width:127px;">Dissociation</td>
			<td>Complete</td>
		</tr>
		<tr>
			<td height="20" style="height:20px;">Base Medium</td>
			<td>419.25 mL</td>
			<td>395.40 mL</td>
			<td>297.00 mL</td>
			<td>178.00 mL</td>
		</tr>
		<tr>
			<td height="20" style="height:20px;width:163px;">PSF*</td>
			<td>5.00 mL</td>
			<td>4.00 mL</td>
			<td>3.00 mL</td>
			<td>2.00 mL</td>
		</tr>
		<tr>
			<td height="40" style="height:40px;width:163px;">Gentamicin Sulfate**</td>
			<td>0.75 mL</td>
			<td>0.60 mL</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td height="40" style="height:40px;width:163px;">Donor Equine Serum</td>
			<td>75.00 mL</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td height="40" style="height:40px;width:163px;">Fetal Bovine Serum***</td>
			<td style="width:155px;">-</td>
			<td>-</td>
			<td>-</td>
			<td>20.00 mL</td>
		</tr>
		<tr>
			<td colspan="4" height="19" style="height:19px;width:660px;">* PSF: penicillin/streptomycin/fungizone cocktail (100x); ** 50 mg/mL concentration; *** Characterized</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 5. Recommended Dilutions and Plating Volumes</td>
		</tr>
		<tr>
			<td height="24" style="height:24px;width:75px;">Plate Size</td>
			<td style="width:103px;">cm^2 per Well</td>
			<td style="width:145px;">Dilution</td>
			<td style="width:111px;">Plating Volume</td>
		</tr>
		<tr>
			<td height="20" style="height:20px;">6 well</td>
			<td>9.5</td>
			<td>1.5-2.0x10^6 cells/mL</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td height="20" style="height:20px;width:75px;">24 well</td>
			<td>1.9</td>
			<td>1.5-2.0x10^6 cells/mL</td>
			<td>250 &#956;L</td>
		</tr>
		<tr>
			<td height="20" style="height:20px;width:75px;">48 well</td>
			<td>0.95</td>
			<td>1.5-2.0x10^6 cells/mL</td>
			<td>150 &#956;L</td>
		</tr>
		<tr>
			<td height="20" style="height:20px;width:75px;">96 well</td>
			<td>0.32</td>
			<td>1.5-2.0x10^6 cells/mL</td>
			<td>50-100 &#956;L</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 6. Average number of cells per g tissue</td>
		</tr>
		<tr>
			<td colspan="2" height="24" style="height:24px;width:201px;">Species</td>
			<td colspan="2" style="width:273px;">Average # cells/g tissue</td>
		</tr>
		<tr>
			<td colspan="2" height="20" style="height:20px;">Danio rerio</td>
			<td colspan="2">6,400,000</td>
		</tr>
		<tr>
			<td colspan="2" height="20" style="height:20px;width:201px;">Danio dangila</td>
			<td colspan="2">1,783,000</td>
		</tr>
		<tr>
			<td colspan="2" height="20" style="height:20px;width:201px;">Devario aequipinnatus</td>
			<td colspan="2">1,797,000</td>
		</tr>
		<tr>
			<td colspan="2" height="20" style="height:20px;">Oncorhynchus mykiss</td>
			<td colspan="2">66,800</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 7. Recommended Incubation Temperatures</td>
		</tr>
		<tr>
			<td colspan="2" height="24" style="height:24px;width:201px;">Species</td>
			<td colspan="2" style="width:273px;">Temperature</td>
		</tr>
		<tr>
			<td colspan="2" height="20" style="height:20px;">Danio/ Devario spp.</td>
			<td colspan="2">26 - 28 &#176;C</td>
		</tr>
		<tr>
			<td colspan="2" height="20" style="height:20px;width:201px;">Oncorhynchus/Salmo spp.</td>
			<td colspan="2">10* - 18 &#176;C</td>
		</tr>
		<tr>
			<td colspan="2" height="20" style="height:20px;width:201px;">Ambystoma mexicanum</td>
			<td colspan="2">18&#176;C</td>
		</tr>
		<tr>
			<td colspan="3" height="20" style="height:20px;">* Lower temperatures support lower proliferation rates.</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of primary myogenic precursor cell/myoblast cultures from basal vertebrate lineages

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e.&#160;teleost fish) and full regeneration following appendage loss (i.e.&#160;urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4.

Twenty-four hours post-seeding, myogenic precursor cells (MPCs) should be visible attached to the laminin substratum (see Figures 1a and 1d). Following seeding, cells (MPCs) adopt a spindle-like shape, indicative of this cell type (Figure 1) and are MyoD1+ (Figure 2). In Danio species, MPCs appear to be more compact with smaller bipolar processes than do MPCs from Oncorhynchus and Salmo species. However, over four days of cultu...

Watch this Scientific Journal Video about Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages at JoVE.com

Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

In vitro culture systems have proven indispensible to our understanding of vertebrate myogenesis. However, much remains to be learned about nonmammalian skeletal muscle development and growth, particularly in basal taxa. An efficient and robust protocol for isolating the adult stem cells of this tissue, the myogenic precursor cells (MPCs), and maintaining their self-renewal, proliferation, and differentiation in a primary culture setting allows for the identification of conserved and divergent regulatory mechanisms throughout the vertebrate lineages.

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem ...

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20.1K Views.  University of Alabama at Birmingham. In vitro culture systems have proven indispensible to our understanding of vertebrate myogenesis. However, much remains to be learned about nonmammalian skeletal muscle development and growth, particularly in basal taxa. An efficient and robust protocol for isolating the adult stem cells of this tissue, the myogenic precursor cells (MPCs), and maintaining their self-renewal, proliferation, and differentiation in a primary culture setting allows for the identifica...

Video: Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

Preparation of Dissociated Mouse Cortical Neuron Cultures

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation. These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation. The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared. Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells. To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step. This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death. Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons.

This video shows a procedure for generating neuronal cultures from late embryo and early postnatal mouse cortex. These cultures can be used for immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging and provide a platform to study the neuronal development of transgenic animals that carry a postnatal lethal gene mutation.

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures ...

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Fabrication of Myogenic Engineered Tissue Constructs

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to the restrictions imposed by a child's small size and their inevitable growth. Consequently, there is a genuine need for innovative therapies designed specifically for pediatric patients with cardiac rhythm disorders. We propose that a conductive biological alternative consisting of a collagen-based matrix containing autologously-derived cells could better adapt to growth, reduce the need for recurrent surgeries, and greatly improve the quality of life for these patients. In the present study, we describe a procedure for incorporating primary skeletal myoblast cell cultures within a hydrogel matrix to fashion a surgically-implantable tissue construct that will serve as an electrical conduit between the upper and lower chambers of the heart. Ultimately, we anticipate using this type of engineered tissue to restore atrioventricular electrical conduction in children with complete heart block. In view of that, we isolate myoblasts from the skeletal muscles of neonatal Lewis rats and plate them onto laminin-coated tissue culture dishes using a modified version of established protocols[2, 3]. After one to two days, cultured cells are collected and mixed with antibiotics, type 1 collagen, Matrigel&trade;, and NaHCO3. The result is a viscous, uniform solution that can be cast into a mold of nearly any shape and size[1, 4, 5]. For our tissue constructs, we employ type 1 collagen isolated from fetal lamb skin using standard procedures[6]. Once the tissue has solidified at 37&deg;C, culture media is carefully added to the plate until the construct is submerged. The engineered tissue is then allowed to further condense through dehydration for 2 more days, at which point it is ready for in vitro assessment or surgical-implantation.

Here, we demonstrate fabrication of collagen-based, tissue constructs containing skeletal myoblasts. These 3-D engineered constructs may be used to replace or repair tissues in vivo. For our purposes, we have designed these as an atrioventricular electrical conduit for the repair of complete heart block[1].

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to ...

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. The large airways therefore require an efficient repair mechanism to protect our bodies. This repair process occurs from stem cells in the airways and isolating these stem cells from the airways is important for understanding the mechanisms of repair and regeneration. It is also important for understanding abnormal repair that can lead to airway diseases 2. The goal of this method is to isolate a novel stem cell population from the mouse tracheal submucosal gland ducts and to place these cells in in vitro and in vivo model systems to identify the mechanisms of repair and regeneration of the submucosal glands 3. This production shows methods that can be used to isolate and assay the duct and basal stem cells from the large airways 3.This will allow us to study diseases of the airway, such as cystic fibrosis, asthma and chronic obstructive pulmonary disease. Currently, there are no methods for isolation of submucosal gland duct cells and there are no in vivo models to study the regeneration of submucosal glands.

Here we demonstrate our protocol for isolation of basal and submucosal gland duct cells from mouse tracheas. We also demonstrate the method of injecting stem cells into the dorsal mouse fat pad to create an in vivo model of submucosal gland regeneration.

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...