This article was supported by NSERC Grant # 299280, IRSST as a scholarship to G. Parent and FRSQ as an undergraduate student research award to M. Cyr. We thank Yoan Lemieux-Laneville for performing the manipulations recorded on video.

Prior to any manipulation, you must identify the group of tendons to be used depending on the experiment you are conducting and the apparatus at your disposition. For our purposes, ventral tendons were chosen because they are smaller and thus easier to manipulate when measuring the cross-sectional area and fitting them into the bioreactor chamber.
Please note that all instruments are autoclaved or sterilized with 70% ethanol. Moreover, a spray bottle containing 70% ethanol is placed beside each work station to sterilize the experimenter&#8217;s gloves before each operation.
Part 1: Extraction
After resection, the tail is carefully manipulated by its extremities to avoid damaging the tissues. Also, to conserve cell viability, all manipulations are carried out in cold saline solution.
1A) Materials:
<ul>
 <li>Cold saline solution (D-PBS)</li>
 <li>Crushed ice</li>
 <li>Surface protector</li>
 <li>Cutting board</li>
 <li>Individual manipulation plates</li>
 <li>2 500 ml dishes</li>
 <li>2 2L glass dishes</li>
 <li>Adhesive tape</li>
 <li>1 Tweezers</li>
 <li>1 Forceps</li>
 <li>1 Tweezers stand</li>
 <li>1 Pair of surgical shears</li>
 <li>1 Scalpel</li>
 <li>1 Pair of surgical scissors 
 
 <img src="/files/ftp_upload/2176/2176fig1.jpg" alt="Figure 1" /> 
 Figure 1. Individual manipulation with orientation identification ("P" for "proximal")</li>
</ul>
1B) Work station:
<ol>
 <li>Spread out the surface protector and place the cutting board on top.</li>
 <li>Fill each two larger glass dishes to halfway with crushed ice and place them on the paper.</li>
 <li>Stick a piece of tape on a corner of the manipulation plate and identify with a letter to indicate the proximal end. </li>
 <li>Fill the two small glass dishes to halfway and all grooves of the manipulation plates with cold saline solution. Place them in the large glass dishes containing ice.</li>
 <li>Organize all the sterile instruments on the surface protector.</li>
 <li>Transfer the tail into one of the glass dish containing saline solution.</li>
</ol>
1C) Manipulations:
<ol>
 <li>Observe the anatomy at the proximal end of the tail to distinguish the ventral side from the dorsal side. The ventral side has a larger number of tendons and a blood vessel that can be verified by gently squeezing the tail to produce tiny drops of blood.</li>
 <li>Using the surgical scissors, cut the skin along the dorsal side of the tail if you want to extract the ventral tendons (or along the ventral side if you need the dorsal tendons). </li>
 <li>Open the incision at the proximal end and remove the skin using forceps or your fingers and by carefully manipulating the tail ends.</li>
 <li>Rinse off the blood and hair in the saline solution and transfer the specimen into the remaining glass dish containing fresh solution. Change gloves. 
 
 A transversal section viewed under light microscopy shows the six tendon groups. One tendon from each group is attached to the caudal end of each vertebra, so we cut at the intervertebral disc to expose new tendons along the cut. 
 
 <img src="/files/ftp_upload/2176/2176fig2.jpg" alt="Figure 2" /> 
 Figure 2. Transversal section of the rat tail viewed under light microscopy.</li>
 <li>Cut the distal end through the cartilage 2-3 vertebrae shorter with surgical shears. Place the tail on the cutting board to cut the soft tissues using a scalpel and then immediately replace it in the solution. </li>
 <li>Using tweezers, pull out one tendon from the distal tail end.</li>
 <li>With a gentle hold at both ends, place the tendon into the individual manipulation plate. Repeat the last two steps until no more tendons can be teased out and cut one vertebra shorter whenever new tissues need to be exposed.</li>
</ol>
Part 2: Cross-sectional area measurement7
When the tissue undergoes mechanical characterization or stimulation, its mechanical properties are described by normalizing the force inside the tendon to stress. This is why we evaluate cross-sectional area.
2A) Materials:
<ul>
 <li>Optic Micrometer</li>
 <li>Stereomicroscope</li>
 <li>Digital camera</li>
 <li>Edge recognition and Profile reconstruction algorithms 5,6</li>
 <li>Cold saline solution (D-PBS)</li>
 <li>1 20-200&mu;L Micro volume pipette</li>
 <li>1 Hex key for optic micrometer adjustments</li>
 <li>2 Tweezers 
 
 <img src="/files/ftp_upload/2176/2176fig3.jpg" alt="Figure 3" /> 
 Figure 3. Optic Micrometer</li>
</ul>
2B) Work station:
<ol>
 <li>Open the software (Digital camera and algorithms).</li>
 <li>Place the optic micrometer so that there is enough room to load the tendon in the apparatus.</li>
 <li>Organize the instruments and glass dish containing the extracted tendons close at hand.</li>
</ol>
2C) Manipulations:
<ol>
	<li>Fill the measuring compartment with cold saline solution until both anchor systems are submerged.</li>
	<li>Transfer the tendon into the compartment by holding the ends with tweezers.</li>
	<li>Using suction via the micro-volume pipette, pull one extremity at a time into the silicon tube. Aspirate until the end extends beyond the shaft collars.</li>
	<li>Tighten the shaft collars to compress the tubes and install the apparatus under the stereomicroscope.</li>
	<li>At 105X magnification, stretch the tendons while observing the tissue until the creases are no longer perceptible. Stretch by additional 0.40%. 
 <img src="/files/ftp_upload/2176/2176fig4.jpg" alt="Figure 4" /> 
 Figure 4. Tendon projection at 105x magnification before and after stretching.</li>
	<li>Adjust position and focus to maintain a clear image of the tendon over a 180&deg; rotation.</li>
	<li>Take a photo at every 10&deg; rotation without modifying tension, focus or position. Use only the rotary shaft to carry out the rotation.
 Due to its cone shape, these steps may be repeated at different points along the tendon.</li>
 <li>Activate the edge recognition and profile reconstruction algorithms in order to analyse the database you have registered.</li>
 <li>Free the tendon by loosening the shaft collars and blowing air into the silicone tubes using the micro volume pipette.</li>
 <li>Reinsert the tendon onto the manipulation plate, again handling it by the ends.</li>
 <li>Verify the shape of the profile reconstruction obtained and the value of the cross-sectional area.</li>
</ol>
Part 3: Rinsing
To remove contamination that may have occurred during the previous manipulations, the tissues are rinsed under biosafety cabinet.
3A) Materials:
<ul>
 <li>Cold saline solution (D-PBS)</li>
 <li>Multiple-groove manipulation plate</li>
 <li>Individual Manipulation plates</li>
 <li>Adhesive tape</li>
 <li>2 Tweezers</li>
 <li>1 Tweezers stand</li>
</ul>
3B) Work station:
<ol>
 <li>Turn on the fan in the biosafety cabinet 15 minutes in advance and clean the inside surfaces with 70% ethanol.</li>
 <li>Bring all the instruments inside the biosafety cabinet.</li>
 <li>Stick pieces of tape onto a corner of each individual plate to identify the proximal end.</li>
 <li>Fill all grooves with sterile saline solution.</li>
</ol>
3C) Manipulations:
<ol>
 <li>Introduce the manipulation plate containing the extracted tendon in the cabinet.</li>
 <li>Using tweezers, remove the tendon from its plate with a gentle grasp on each end.</li>
 <li>Immerse and gently stir the tendon in the saline solution of each compartment of the multiple-groove manipulation plate, starting from the closest and proceeding to the furthest groove.</li>
 <li>Finally, submerge the tendon in an individual manipulation plate. </li>
</ol>
Part 4: Loading into bioreactor chamber
To avoid further contamination, the following manipulations are also conducted in the biosafety cabinet.
4A) Materials:
<ul>
 <li>Bioreactor chamber 4</li>
 <li>Sterile anchors</li>
 <li>1 Sterile Petri dish</li>
 <li>Cold cell cultured Medium (DMEM)</li>
 <li>Cyanoacrylate</li>
 <li>2 Tweezers</li>
 <li>Tweezers stand</li>
 <li>0.5-10&mu;l Micro-volume pipette</li>
 <li>1 Ruler 
 
 <img src="/files/ftp_upload/2176/2176fig5.jpg" alt="Figure 5" /> 
 Figure 5. Bioreactor chamber</li>
</ul>
4B) Work station:
<ol>
 <li>Since the previous step was also realized in biosafety cabinet, removal of unnecessary tools will give you more work space. </li>
 <li>Bring all other instruments inside the cabinet.</li>
</ol>
4C) Manipulations:
<ol>
 <li>Place an anchor on the manipulation plate with its spool centered over the tendon extremity and wound the tissue up and around the spool.</li>
 <li>Roll the anchor over and hold the loose extremity while ensuring that the tissue stays attached to the anchor. Stop after &frac34; turn. </li>
 <li>Carry out the same steps with the other end.</li>
 <li>Roll both anchors over equally until there is 6 cm between their centers1. </li>
 <li>Loosen the tendon to immerse it in the solution and allow the anchored sections to dry briefly to enhance its mechanical properties2-3.</li>
 <li>Pour cyanoacrylate into the Petri dish and draw 2.5&mu;L into the micro volume pipette.</li>
 <li>Apply a drop of cyanoacrylate to the wound-up tissue without dropping any glue in the saline solution. Repeat until 2 drops have been applied to each anchor.</li>
 <li>With the tendon still completely immersed, wait 5 minutes for the glue to dry completely.</li>
 <li>During this time, fill the chamber compartment with saline solution.</li>
 <li>Rehydrate all tissue with saline solution to prevent damage.</li>
 <li>By manipulating only the anchors, transfer the tendon into the bioreactor chamber and close it to prevent leakage and contamination during transfer.</li>
</ol>
Part 5: Representative Results:
The outcome of the protocol shows that when performed correctly, our tissue rigorous isolation and preparation procedure make it possible to maintain tissue sterility, viability and integrity.
First, using the simple and repetitive extraction method, we are able to extract tail tendons without damaging the collagen network as it can be observed by a microscopic analysis of H&amp;E tainted sections realized after the extraction.
<img src="/files/ftp_upload/2176/2176fig6.jpg" alt="Figure 6" /> 
 Figure 6. Tendon collagen network after extraction (5&mu;m longitudinal section stained with H&amp;E).
We then cultured tendons for up to ten days and conducted sterility tests. Each day, we plated used culture solution on agar and incubated it for 24 hours. Since no bacterial growth was observed, we concluded that our manipulations do not result in contamination.
With the profile reconstruction algorithm and optic micrometer, we are able to estimate the cross-sectional area within a 2% margin of error7.
<img src="/files/ftp_upload/2176/2176fig7.jpg" alt="Figure 7" /> 
 Figure 7. Profile reconstruction of a RTT.
Finally, we assessed viability using the LIVE/DEAD Viability/Cytotoxicity Kit for mammalian cells after the rinsing step and after a twelve-day culture period. Since a large majority of green fluorescent live cells were evident, we can confirm that our isolation procedures are successful in preserving live tissue. The same test was performed two hours after attaching the tendon into the bioreactor chamber. We verified that the dehydration and glue at the anchor had not spread in the tissue between both anchors.
<img src="/files/ftp_upload/2176/2176fig8.jpg" alt="Figure 8" /> 
 Figure 8. Tissue viability at the anchor (green = live cells, red = dead cells)

<ol>
	<li>Bennett, M. B., Ker, R., Dimery, N., Alexander, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+various+mammalian+tendons.">Mechanical properties of various mammalian tendons.</a> J. Zool. Lond. A209, 537-548 (1986).</li><li>Dupuis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> D&#233;shydratation de matrices collag&#233;niques reconstruites in vitro: effets sur les propri&#233;t&#233;s m&#233;caniques et histologiques [dissertation]. , 114-114 (1981).</li><li>Haut, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+influence+of+strain+rate+on+the+tensile+failure+of+rat+tail+tendon.">Age-dependent influence of strain rate on the tensile failure of rat tail tendon.</a> Journal of Biomechanical Engineering. 105, 296-299 (1983).</li><li>Hupp&#233;, N., Lessard, J. -. L., Langelier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Bioreactor+Design+for+the+Mechanobiological+Study+of+Soft+Tissue+Damage+and+Repair+in+Conditions+that+Provide+the+Best+Approximation+of+Actual+Use.">A Bioreactor Design for the Mechanobiological Study of Soft Tissue Damage and Repair in Conditions that Provide the Best Approximation of Actual Use.</a> , (1985).</li><li>Langelier, E., Dupuis, D., Guillot, M., Goulet, F. &. a. m. p. ;. a. m. p., Rancourt, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-sectional+profiles+and+volume+reconstructions+of+soft+tissues+using+laser+beam+measurements.">Cross-sectional profiles and volume reconstructions of soft tissues using laser beam measurements.</a> J Biomech Eng. 126, 796-802 (2004).</li><li>Lee, T. Q., Woo, S. L. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+determining+cross-sectional+shape+and+area+of+soft+tissues.">A new method for determining cross-sectional shape and area of soft tissues.</a> J Biomech Eng. 110, 110-114 (1988).</li><li>Parent, G., Cyr, M., Cousineau-Pelletier, P., Desbiens-Blais, F. &. a. m. p. ;. a. m. p., Langelier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+Techniques+for+the+Evaluation+of+Cross-Sectional+Area+in+Rat+Tail+Tendons+Generate+Significant+Errors.">Current Techniques for the Evaluation of Cross-Sectional Area in Rat Tail Tendons Generate Significant Errors.</a> , (1985).</li></ol>

No conflicts of interest declared.

By applying those experimental procedures, we can conduct a wide variety of in vitro studies on this kind of tissues. As for example, a study on tissue degeneration was carried out by the application of under-stimulation to RTTs for a period of ten days. Each day, we evaluated tissue mechanical properties in non-destructive stress relaxation tests. At the end, we were able to observe RTT stress variation and thus analyze the progression of mechanical properties.
<img src="/files/ftp_...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>D-PBS </td>
<td>Reagent</td>
<td>Wisent inc.</td>
<td>311-410-CL</td>
<td>Saline solution</td>
</tr>
<tr>
<td>Glucose </td>
<td>Reagent</td>
<td>Wisent inc. </td>
<td>609-037-EL </td>
<td>Saline solution
 1g/L</td>
</tr>
<tr>
<td>Antibiotics-antimycotics </td>
<td>Reagent</td>
<td>Invitrogen Corp.</td>
<td>15240-062 </td>
<td>Saline solution
 1%</td>
</tr>
<tr>
<td>DMEM</td>
<td>Reagent</td>
<td>Invitrogen Corp.</td>
<td>12800-017</td>
<td>Culture solution</td>
</tr>
<tr>
<td>Sodium Bicarbonate</td>
<td>Reagent</td>
<td>Wisent inc.</td>
<td>600-105-CG</td>
<td>Culture solution 3.7g/L</td>
</tr>
<tr>
<td>FBS</td>
<td>Reagent</td>
<td>Wisent inc.</td>
<td>090150</td>
<td>Culture solution 10%</td>
</tr>
<tr>
<td>Antibiotics-antimycotics</td>
<td>Reagent</td>
<td>Invitrogen Corp.</td>
<td>15240-062</td>
<td>Culture solution 1%</td>
</tr>
<tr>
<td>Optic Micrometer</td>
<td>Tool</td>
<td>Custom-made</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Manipulation plate</td>
<td>Tool</td>
<td>Custom-made</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Bioreactor chamber</td>
<td>Tool</td>
<td>Custom-made</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

preparation of rat tail tendons for biomechanical and mechanobiological studies

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working with those tissues is challenging because they are very fragile, and until now there was no rigorously detailed protocol for their isolation.
Faced with these challenges, we have developed methods and instruments to facilitate manipulation of RTTs and control tissue viability, sterility and integrity. This article describes the experimental procedures used to prepare RTTs for biomechanical and mechanobiological studies. Our work is divided into four main steps: extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.
At each step, all procedures, materials and manipulations are presented in detail so that they can be easily reproduced. Moreover, the specific instruments developed are presented: a manipulation plate used to segregate RTTs, an optic micrometer to position the tissue during the cross-sectional area measurement and an anchoring system to attach the RTTs onto a bioreactor.
Finally, we describe the results obtained after multiple tests to validate our methods. The viability, sterility and integrity evaluations demonstrate that our procedures are sufficiently rigorous for manipulations of fragile tissues such as rat tail tendons.

Watch this Scientific Journal Video about Preparation of Rat Tail Tendons for Biomechanical and Mechanobiological Studies at JoVE.com

Preparation of Rat Tail Tendons for Biomechanical and Mechanobiological Studies

This article describes the experimental procedures used to prepare rat tail tendons for biomechanical and mechanobiological studies. Several features of the main steps in preparation are demonstrated, beginning with extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working ...

preparation-rat-tail-tendons-for-biomechanical-mechanobiological

Research

JoVE Journal

Biology

16.8K Views.  Université de Sherbrooke. This article describes the experimental procedures used to prepare rat tail tendons for biomechanical and mechanobiological studies. Several features of the main steps in preparation are demonstrated, beginning with extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.

Video: Preparation of Rat Tail Tendons for Biomechanical and Mechanobiological Studies

Interview: Protein Folding and Studies of Neurodegenerative Diseases

In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the protein folding is at the core of the modern biology. In addition to their traditional biochemical functions, proteins can mediate transfer of biological information and therefore can be considered a genetic material. This recently discovered function of proteins has important implications for studies of human disorders. Dr. Lindquist also describes current experimental approaches to investigate the mechanism of neurodegenerative diseases based on genetic studies in model organisms.

In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the protein folding is at the core of the modern biology. In addition to their traditional biochemical functions, proteins can mediate transfer of biological information and therefore can be considered a genetic material. This recently discovered function of proteins has important implications for studies of human disorders. Dr. Lindquist also describes current experimental approaches to investigate the mechanism of neurodegenerative diseases based on genetic studies in model organisms.

In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the ...

Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) would provide a powerful platform for neuroscientists to investigate axo-glial interactions, properties and differentiation of multipotent progenitors, and progression of oligodendroglial lineage cells at the cellular and molecular level.  We describe here a CNS aggregate culture system from embryonic rat forebrains, which can be maintained in a serum-free medium up to 3-4 weeks and is used in our laboratory as a model to study neuron-glia interaction and CNS myelination.  This video clip will demonstrate how to isolate and grow these CNS aggregate cultures from E16 rat brain.  Furthermore, from the same brain dissection, highly enriched regular dissociated neuronal cultures can be readily obtained and used for various studies on CNS neurons or used for co-cultures with other cells. 

A protocols for an embryonic rat brain aggregate culture system is described. Multipotent progenitors in the aggregates can develop and differentiate into neurons, astrocytes and oligodendrocytes.

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) ...

Preparation and Culture of Rat Lens Epithelial Explants for Studying Terminal Differentiation

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  Following division, these cells migrate posteriorly, where FGF diffusing from the retina induces them to differentiate into a posterior array of elongated lens fiber cells, which compose the bulk of the lens.  Differentiation of lens epithelial cells into lens fibers can be induced in vitro by culturing explants of the central region of the anterior epithelium in the presence of FGF-2.  Explants are prepared from lenses of neonatal rats by removing the lens from the eye and grasping the lens capsule on the posterior side with dissecting tweezers.  The posterior capsule is then gently torn open and pressed down into the plastic bottom of a tissue culture dish. The peripheral regions of the explant are removed with a scalpel and the central area is then cultured in the presence of 100ng/ml FGF-2 for as long as 2-3 weeks, depending on the parameters to be studied.  Since epithelial cells in cultured explants differentiate in approximate synchrony over a period of days to weeks, the time course of signaling and gene expression can be determined using molecular, biochemical, and pharmacological techniques.  Immunofluorescence microscopy is a powerful adjunct to these methods as it demonstrates the subcellular localization of proteins of interest and can reveal the physiological consequences of experimental manipulations of signaling pathways.

Explants of the central region of rat lens epithelia differentiate synchronously when cultured in the presence of FGF-2. Immunofluorescence microscopy of such cultures can provides novel information about gene expression and signaling events associated with terminal differentiation.

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Preparation of Highly Coupled Rat Heart Mitochondria

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there have been significant advances in our understanding of the functions of mitochondria other than the generation of energy. These include their role in apoptosis, acting as signalling organelles, mammalian development and ageing as well as their contribution to the coordination between cell metabolism and cell proliferation. Our understanding of biological processes modulated by mitochondria is based on robust methods for isolation and handling of intact mitochondria from tissues of the laboratory animals. Mitochondria from rat heart is one of the most common preparations for past and current studies of cellular metabolism including studies on knock-out animals.
Here we describe a detailed rapid method for isolation of intact mitochondria with a high degree of coupling. Such preparation of rat heart mitochondria is an excellent object for functional and structural research on cellular bioenergetics, transport of biomolecules, proteomic studies and analysis of mitochondrial DNA, proteins and lipids.

We describe &#1072; protocol for isolation of pure, highly coupled rat heart mitochondria for functional or structural studies of cellular bioenergetics, biophysical measurements, proteomics or mitochondrial DNA and lipids analysis.

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe a new strategy that combines the use of chemical epigenetic erasing with mechanosensing-related cues, to generate mammalian pluripotent cells. Two main steps are required. In the first step, adult mature (terminally differentiated) cells are exposed to the epigenetic eraser 5-aza-cytidine to drive them into a pluripotent state. This part of the protocol was developed, based on the increasing understanding of the epigenetic mechanisms controlling cell fate and differentiation, and involves the use of the epigenetic modifier to erase cell differentiated state and then drive into a transient high plasticity window.
In the second step, erased cells are encapsulated in polytetrafluoroethylene (PTFE) micro-bioreactors, also known as Liquid Marbles, to promote 3D cell rearrangement to extend and stably maintain the acquired high plasticity. PTFE is a non-reactive hydrophobic synthetic compound and its use permits the creation of a cellular microenvironment, which cannot be achieved in traditional 2D culture systems. This system encourages and boosts the maintenance of pluripotency though bio-mechanosensing-related cues.
The technical procedures described here are simple strategies to allow for the induction and maintenance of a high plasticity state in adult somatic cells. The protocol allowed the derivation of high plasticity cells in all mammalian species tested. Since it does not involve the use of gene transfection and is free of viral vectors, it may represent a notable technological advance for translational medicine applications. Furthermore, the micro-bioreactor system provides a notable advancement in stem cell organoid technology by in vitro re-creating a specific micro-environment that allows for the long-term culture of high plasticity cells, namely as ESCs, iPSCs, epigenetically erased cells and MSCs.

We here present a method that combines the use of chemical epigenetic erasing with mechanosensing-related cues to efficiently generate mammalian pluripotent cells, without the need of gene transfection or retroviral vectors. This strategy is, therefore, promising for translational medicine and represents a notable advancement in stem cell organoid technology.

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe ...

Cryo-Electron Microscopic Grid Preparation for Time-Resolved Studies using a Novel Robotic System, Spotiton

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental challenge of great interest to the field of cryo-electron microscopy (cryo-EM). A few methodological strategies have been developed that support these &#34;time-resolved&#34; studies, one of which, Spotiton-a novel robotic system-combines the dispensing of picoliter-sized sample droplets with precise temporal and spatial control. The time-resolved Spotiton workflow offers a uniquely efficient approach to interrogate early structural rearrangements from minimal sample volume. Fired from independently controlled piezoelectric dispensers, two samples land and rapidly mix on a nanowire EM grid as it plunges toward the cryogen. Potentially hundreds of grids can be prepared in rapid succession from only a few microliters of a sample. Here, a detailed step-by-step protocol of the operation of the Spotiton system is presented with a focus on troubleshooting specific problems that arise during grid preparation.

The protocol presented here describes the use of Spotiton, a novel robotic system, to deliver two samples of interest onto a self-wicking, nanowire grid that mix for a minimum of 90 ms prior to vitrification in liquid cryogen.