The authors would like to acknowledge John Woolford for the initial basis of this method, TGK is supported by NIH GM057483 and AI076245 and PRC is supported by GM77073.

1. Preparation of 7-47% sucrose gradients
<ol>
	<li>Prepare sucrose gradients one day before use to allow gradients to become continuous. Make sterile 7% and 47% sucrose solutions containing 50mM NH4Cl, 50mM Tris-Acetate pH 7.0 and 12mM MgCl2 and store at 4&deg;C 1.</li>
	<li>For 6 gradients mix the 7% and 47% sucrose stock solutions to make 48 mL of each of the following sucrose concentrations. Add DTT (1M stock) to each sucrose solution to a final concentration of 1mM. 
<table border="1" cellspacing="0" cellpadding="0">
<tbody>
 <tr>
 <td>Final Concentration</td>
 <td>7% sucrose</td>
 <td>47% sucrose</td>
 </tr>
 <tr>
 <td>7%</td>
 <td>48 mL</td>
 <td>0</td>
 </tr>
 <tr>
 <td>17%</td>
 <td>36 mL</td>
 <td>12 mL</td>
 </tr>
 <tr>
 <td>27%</td>
 <td>24 mL</td>
 <td>24 mL</td>
 </tr>
 <tr>
 <td>37%</td>
 <td>12 mL</td>
 <td>36 mL</td>
 </tr>
 <tr>
 <td>47%</td>
 <td>0</td>
 <td>48 mL</td>
 </tr>
</tbody>
</table></li>
	<li>In order to pour the gradients, attach a Pasteur pipette to a 20 mL syringe by securing and sealing with Parafilm or use a long needle. Add 7 mL of 7% sucrose to the bottom of a 1 x 3.5" polyallomer ultracentrifuge tube. Next add 7 mL of 17% sucrose solution under the 7% solution by placing the Pasteur pipette tip near bottom of tube and pipetting no faster than 0.3 mL/sec. Repeat with 7 mL each of 27%, 37% and 47% sucrose solutions. You should observe clear lines between the layers, indicating that minimal mixing has occurred. Store gradients at 4&deg;C overnight along with the rotors, bottles and tubes that will be used to harvest samples.</li>
</ol>
2. Preparation of the yeast extract
<ol>
	<li>Grow 125 mL of yeast culture to an OD600 of 0.8-1.0. Add cycloheximide to the culture to a final concentration of 100 &mu;g/mL and continue shaking at 30&deg;C for 15 min.</li>
	<li>Meanwhile, prepare lysis buffer containing 80 &mu;g/mL cycloheximide, 200 &mu;g/mL heparin, 0.2% DEPC, and 10 mM Tris-HCl pH 7.5, 0.1M NaCl, 30mM MgCl2. Keep everything on ice from this point on.</li>
	<li>Pour the culture into a 250 mL centrifuge bottle and fill with ice. Centrifuge at 8,000 x g (7,250 rpm in a SLA-1500 rotor) for 5 min at 4&deg;C. Wash the pellet once with 10 mL of ice cold lysis buffer and transfer to 15 mL polypropylene tubes. Centrifuge at 5,900 x g (~6,000rpm in a SLA-600TC rotor) for 3 min at 4&deg;C. Resuspend the pellet in 0.5 mL of lysis buffer, transfer to 1.5mL tube and add 0.5 mL of chilled, baked acid-washed glass beads. Vortex the cells for four 30 sec intervals, cooling the tube on ice for 30 sec between each interval. Add another 0.5 mL of lysis buffer into the 1.5 mL tube. Centrifuge at maximum speed in a microcentrifuge (14,000 rpm in an Eppendorf 5417R microcentrifuge) for 10 min at 4&deg;C. Transfer supernatant to a new 1.5 mL microcentrifuge tube. Remove 10 &mu;l to a separate tube to determine the OD260 / OD280 ratio (see step 3.1 below). Store extracts at -80&deg;C.</li>
</ol>
3. Preparation of extracts from mammalian cells
<ol>
	<li>For adherent cells, grow in 100 mm dish to ~70% confluence. You will need one 100 mm dish per 11 ml gradient (typical yield is ~20 OD260 units). Scale the amount linearly for larger or smaller gradient sizes. For non-adherent cells, approximately 1 x 107 cells are needed per 11 ml gradient.</li>
	<li>Prior to lysis, add cycloheximide to 100 &mu;g/mL. Incubate at 37&deg;C for 15 min. Transfer plates to ice and rinse cells twice in ice cold PBS. All future steps MUST be carried out at 0-4&deg;C.</li>
	<li>Remove all traces of PBS by aspiration (let plates drain on an angled bed of ice) and add 1 mL lysis buffer, scrape and transfer to pre-chilled 1.5 mL tube. Lysis buffer components will need to be optimized to the cell type and growth conditions. A good starting buffer would be 20 mM HEPES-KOH, pH 7.4, 15 mM MgCl2, 200 mM KCl, 1% Triton X-100 (v/v), 100 &mu;g/mL cycloheximide, 2 mM DTT, and 1 mg/mL heparin. The key variables are the concentrations of magnesium and potassium chloride as well as the inclusion or exclusion of non-ionic detergents to improve extraction of cytoskeletal or membrane bound polysomes. Lyse cells by passing through 27 gauge syringe needle twice or by 5-10 strokes of dounce homogenization using Type B pestle. The syringe or homogenizer should be pre-chilled prior to use.</li>
	<li>Spin at 14,000 x g for 5 min in refrigerated microcentrifuge. Transfer fresh lysate to the sucrose gradient (for mammalian cells this is made up in lysis buffer lacking Triton X-100 and heparin but otherwise as described in 1.2 and 1.3) or flash freeze in liquid nitrogen for later use.</li>
</ol>
4. Centrifugation of gradients
<ol>
	<li>Thaw polyribosome lysates on ice. Determine the concentration of nucleic acid in the lysate by adding the reserved 10 &mu;L sample to 1 mL of water and reading the OD260/OD280 ratio with a spectrophotometer. Typical yield from a yeast culture grown as described is 50 OD units.</li>
	<li>Using a pipette with the tip placed against the wall of the tube near the surface of the gradient, gently layer 25 OD260 units of the lysate on top of the gradient. For a 35 mL gradient 500 &mu;l of lysate is typically loaded. Lysis buffer can be used to assure all gradients are loaded with an equal volume. Lower amounts of material yield better resolution. Using forceps, carefully lower the gradients into buckets of a swinging bucket rotor.</li>
	<li>Centrifuge the gradients at 100,000 x g (23,000 rpm in a Surespin 630 rotor) for 4 hr at 4&deg;C. This protocol can be adapted for smaller volume gradients.</li>
</ol>
5. Collection of data and fractions
<ol>
	<li>Approximately 30 min prior to the completion of the 4 hr spin attach the needle to the Model 184 tube piercer. Attach the pen to the online Isco Model UA-5 Absorbance/Fluorescence Monitor. Power on the absorbance monitor to allow a stable baseline to be reached prior to analyzing samples. Electronic acquisition of the polysome profile can easily be obtained by attaching a DI-148U data acquisition unit (DATAQ Instruments) to the chart recorder. Profiles can then be collected in real time and saved for later annotation using the accompanying WinDaq software.</li>
	<li>Fill the syringe pump with 50mL of Fluorinert using the reverse flow, rapid setting. Make sure that there are no air bubbles present. Connect the hose from the syringe to the tube piercer and briefly turn on the flow of Fluorinert at 3 mL/min in the forward direction to clear the hose of any air bubbles.</li>
	<li>Place a waste beaker or series of tubes if collecting fractions under the hose at the end of the flow cell to collect sample run off.</li>
	<li>Carefully remove the polyallomer centrifuge tubes containing the sucrose gradient from centrifuge rotor and place them on ice (pre-form the wells in the ice with an empty tube to avoid disturbing gradients).</li>
	<li>To analyze cell extracts, place the gradient tube on the tube piercer. Connect the top of the tube to the outlet and secure. Raise the bottom stage to the centrifuge tube so that the needle pierces the bottom of the tube. Once the needle has protruded through the bottom of the centrifuge tube, begin the flow of Fluorinert in the forward direction at 6mL/min.</li>
	<li>Once the Fluorinert has begun flowing into the tube, monitor the movement of the needle on the online absorbance monitor. Once the needle begins to move, turn on the chart movement to a speed setting of 60. The monitor will record the OD254 starting with the free material followed by 40S ribosomal subunit detection and continuing through to polyribosome complexes. If you are collecting fractions for RNA or protein analysis, typically 0.2 min fractions are taken (1 mL). For RNA analysis, fractions should be collected directly into tubes containing Trizol (Invitrogen), Phenol or SDS/proteinase K, depending on the preferred method of extraction.</li>
	<li>When the end of the polyribosome profile has been reached, turn off the flow of Fluorinert to the centrifuge tube and stop the chart movement of the absorbance/fluorescence monitor.</li>
	<li>Retract the Fluorinert from the centrifuge tube into the syringe by beginning the flow in the reverse direction at a rapid rate making sure not to draw sucrose from the tube into the syringe. Unscrew the centrifuge tube from the tube piercer, lower the needle, and remove the tube.</li>
	<li>Repeat steps 5.5 through 5.8 for each sucrose gradient.</li>
	<li>When all sucrose gradients have been analyzed, retract the Fluorinert from the hose back into the syringe. Remove each part of fractionator (tube piercer and flow cell) including the waste hose located at the top and rinse well with water.</li>
</ol>
6. Representative Results
<img src="/files/ftp_upload/1948/1948fig1.jpg" alt="Figure 1" /> Figure 1. Representative trace of ribosome extract prepared from yeast strain BY4741 (mata his3&Delta;1 leu2&Delta;0 met15&Delta;0 ura3&Delta;0) in the presence of cycloheximide. The ribosomal extract was fractionated utilizing a 7 47% sucrose gradient and analyzed using a pump syringe apparatus attached to a UV detector. Peaks containing polyribosomes and 80S, 60S and 40S ribosomes are indicated.

<ol>
	<li>Baim, S. B., Pietras, D. F., Eustice, D. C., Sherman, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mutation+allowing+an+mRNA+secondary+structure+diminishes+translation+of+Saccharomyces+cerevisiae+iso-1-cytochrome+C.">A mutation allowing an mRNA secondary structure diminishes translation of Saccharomyces cerevisiae iso-1-cytochrome C.</a> Mol. Cell. Biol. 5, 1839-1846 (1985).</li><li>Ripmaster, T. L., Vaughn, G. P., Woolford, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DRS1+to+DRS7,+novel+genes+required+for+ribosome+assembly+and+function+in+Saccharomyces+cerevisiae.">DRS1 to DRS7, novel genes required for ribosome assembly and function in Saccharomyces cerevisiae.</a> Mol. Cell. Biol. 13, 7901-7912 (1993).</li><li>Anand, M., Chakraburtty, K., Marton, M. J., Hinnebusch, A. G., Kinzy, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+interactions+between+yeast+translation+eukaryotic+elongation+factor+(eEF)+1A+and+eEF3.">Functional interactions between yeast translation eukaryotic elongation factor (eEF) 1A and eEF3.</a> J Biol Chem. 278, 6985-6991 (2003).</li><li>Serikawa, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcriptome+and+its+translation+during+recovery+from+cell+cycle+arrest+in+Saccharomyces+cerevisiae.">The transcriptome and its translation during recovery from cell cycle arrest in Saccharomyces cerevisiae.</a> Mol Cell Proteomics. 2, 191-204 (2003).</li><li>Ingolia, N. T., Ghaemmaghami, S., Newman, J. R., Weissman, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+in+vivo+of+translation+with+nucleotide+resolution+using+ribosome+profiling.">Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling.</a> Science. 324, 218-223 (2009).</li><li>Valasek, L., Szamecz, B., Hinnebusch, A. G., Nielsen, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+stabilization+of+preinitiation+complexes+by+formaldehyde+cross-linking.">In vivo stabilization of preinitiation complexes by formaldehyde cross-linking.</a> Methods Enzymol. 429, 163-183 (2007).</li><li>Fletcher, J. E., Copeland, P. R., Driscoll, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome+distribution+of+phospholipid+hydroperoxide+glutathione+peroxidase+mRNA:+evidence+for+a+block+in+elongation+at+the+UGA/selenocysteine+codon.">Polysome distribution of phospholipid hydroperoxide glutathione peroxidase mRNA: evidence for a block in elongation at the UGA/selenocysteine codon.</a> Rna. 6, 1573-1584 (2000).</li><li>Ruan, H. J., Brown, C. Y., Morris, D. R., Richter, J. 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> Analysis of mRNA formation and function. , 305-321 (1997).</li><li>Martin, G. W., Berry, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selenocysteine+codons+decrease+polysome+association+on+endogenous+selenoprotein+mRNAs.">Selenocysteine codons decrease polysome association on endogenous selenoprotein mRNAs.</a> Genes Cells. 6, 121-129 (2001).</li><li>Lee, Y. Y., Cevallos, R. C., Jan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+upstream+open+reading+frame+regulates+translation+of+GADD34+during+cellular+stresses+that+induce+eIF2alpha+phosphorylation.">An upstream open reading frame regulates translation of GADD34 during cellular stresses that induce eIF2alpha phosphorylation.</a> J Biol Chem. 284, 6661-6673 (2009).</li></ol>

No conflicts of interest declared.

The information obtained from the polyribosome profile can provide valuable insight into the translational status of the cell. In addition, the status of the assembly of ribosomal subunits themselves can be determined2. For example the presence of halfmers or 80S and larger peaks with a slight shoulder to the right on the profile indicates a bound 40S subunit awaiting 60S subunit joining. Performing the experiment in the absence of any added cycloheximide or other inhibitor of elongation allows for analysis of the rate of run off, which indicates whether or not elongation is altered3. The fractions themselves are a rich source of reagents for the subsequent determination of the association of a specific mRNA or protein with ribosomal subpopulations by Northern or Western blotting of the fractions. The total pool of mRNAs associated with active ribosomes can be determined via an associated microarray4 or deep sequencing analysis5. The protein associations can also be stabilized by the appropriate addition of a cross linking reagent 6.
Polyribosome analysis from mammalian cells is also worth mentioning as a distinct protocol in terms of the apparent difficulties in establishing the conditions required to obtain stable polysomes. The buffer systems reported in the literature are widely variable 7-10, thus there may be a requirement for cell-type specific optimization of the lysis buffer, or it is equally plausible that the buffer system is not as important as keen attention to working with fresh lysates kept at low temperatures. To our knowledge a systematic evaluation of the parameters affecting mammalian polysome formation has not been reported.

<!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>polyallomer ultracentrifuge tube</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>326823</td>
<td>1 x 3.5 in. (25x89mm)</td>
</tr>
<tr>
<td>Fluorinert</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F9755</td>
<td>&nbsp;</td>
<tr>
<td>Cycloheximde</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C-7698</td>
<td>&nbsp;</td>
<tr>
<td>Heparin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3393</td>
<td>&nbsp;</td>
<tr>
<td>BY4741</td>
<td>&nbsp;</td>
<td>Open Biosystems</td>
<td>YSC1048</td>
<td>Other strains work equally well</td>
</tr>		</tbody>
		</table>
		</div>

eukaryotic polyribosome profile analysis

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase or the elongation phase by a variety of cellular stresses such as amino acid starvation or growth factor withdrawal. Alternatively, translation of individual mRNAs can be regulated by mRNA localization or the presence of cognate microRNAs. Studies of protein synthesis frequently utilize polyribosome analysis to shed light on the mechanisms of translation regulation or defects in protein synthesis. In this assay, mRNA/ribosome complexes are isolated from eukaryotic cells.  A sucrose density gradient separates mRNAs bound to multiple ribosomes known as polyribosomes from mRNAs bound to a single ribosome or monosome. Fractionation of the gradients allows isolation and quantification of the different ribosomal populations and their associated mRNAs or proteins. Differences in the ratio of polyribosomes to monosomes under defined conditions can be indicative of defects in either translation initiation or elongation/termination. Examination of the mRNAs present in the polyribosome fractions can reveal whether the cohort of individual mRNAs being translated changes with experimental conditions. In addition, ribosome assembly can be monitored by analysis of the small and large ribosomal subunit peaks which are also separated by the gradient. In this video, we present a method for the preparation of crude ribosomal extracts from yeast cells, separation of the extract by sucrose gradient and interpretation of the results.  This procedure is readily adaptable to mammalian cells.

Watch this Scientific Journal Video about Eukaryotic Polyribosome Profile Analysis at JoVE.com

Eukaryotic Polyribosome Profile Analysis

This article describes a protocol for the extraction of translating ribosomes from eukaryotic cells. Once extracted, ribosomes are separated into monosomes and polyribosomes by sucrose gradient fractionation to allow different ribosomal populations to be analyzed. As such, this method is the gold standard for examining the regulation of translation.

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase ...

Research

JoVE Journal

Biology

52.5K Views.  University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School. This article describes a protocol for the extraction of translating ribosomes from eukaryotic cells. Once extracted, ribosomes are separated into monosomes and polyribosomes by sucrose gradient fractionation to allow different ribosomal populations to be analyzed. As such, this method is the gold standard for examining the regulation of translation.

Video: Eukaryotic Polyribosome Profile Analysis

Immunoblot Analysis

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. It involves the solubilization and electrophoretic separation of proteins, glycoproteins, or lipopolysaccharides by gel electrophoresis, followed by quantitative transfer and irreversible binding to nitrocellulose, PVDF, or nylon. The immunoblotting technique has been useful in identifying specific antigens recognized by polyclonal or monoclonal antibodies and is highly sensitive (1 ng of antigen can be detected). This unit provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization using chromogenic or chemiluminescent substrates.

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. This video provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization using chromogenic or chemiluminescent substrates.

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the ...

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Bioenergetic Profile Experiment using C2C12 Myoblast Cells

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in understanding the role of mitochondrial function in obesity, diabetes, aging, cancer, cardiovascular function and safety toxicity.
Cellular metabolism is the process of substrate uptake, such as oxygen, glucose, fatty acids, and glutamine, and subsequent energy conversion through a series of enzymatically controlled oxidation and reduction reactions. These intracellular biochemical reactions result in the production of ATP, the release of heat and chemical byproducts, such as lactate and CO2 into the extracellular environment.
Valuable insight into the physiological state of cells, and the alteration of the state of those cells, can be gained through measuring the rate of oxygen consumed by the cells, an indicator of mitochondrial respiration - the Oxygen Consumption Rate - or OCR. Cells also generate ATP through glycolysis, i.e.: the conversion of glucose to lactate, independent of oxygen. In cultured wells, lactate is the primary source of protons. Measuring the lactic acid produced indirectly via protons released into the extracellular medium surrounding the cells, which causes acidification of the medium provides the Extra-Cellular Acidification Rate - or ECAR.
In this experiment, C2C12 myoblast cells are seeded at a given density in Seahorse cell culture plates. The basal oxygen consumption (OCR) and extracellular acidification (ECAR) rates are measured to establish baseline rates. The cells are then metabolically perturbed by three additions of different compounds (in succession) that shift the bioenergetic profile of the cell.
This assay is derived from a classic experiment to assess mitochondria and serves as a framework with which to build more complex experiments aimed at understanding both physiologic and pathophysiologic function of mitochondria and to predict the ability of cells to respond to stress and/or insults.

A description of a method for profiling mitochondrial function in cells is provided. The mitochondrial profile generated provides four parameters of mitochondrial function that can be measured in one experiment: basal respiration rate, ATP-linked respiration, proton leak, and reserve capacity.

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in ...

Correlative Light and Electron Microscopy (CLEM) as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity necessary for proper cellular function. Understanding how the enzymes, proteins, and cytoskeletal components govern and maintain this biochemical segregation is therefore of paramount importance. The use of fluorescently tagged molecules to localize to and/or perturb subcellular compartments has yielded a wealth of knowledge and advanced our understanding of cellular regulation. Imaging techniques such as fluorescent and confocal microscopy make ascertaining the position of a fluorescently tagged small molecule relatively straightforward, however the resolution of very small structures is limited 1.
On the other hand, electron microscopy has revealed details of subcellular morphology at very high resolution, but its static nature makes it difficult to measure highly dynamic processes with precision 2,3. Thus, the combination of light microscopy with electron microscopy of the same sample, termed Correlative Light and Electron Microscopy (CLEM) 4,5, affords the dual advantages of ultrafast fluorescent imaging with the high-resolution of electron microscopy 6. This powerful technique has been implemented to study many aspects of cell biology 5,7. Since its inception, this procedure has increased our ability to distinguish subcellular architectures and morphologies at high resolution. 
Here, we present a streamlined method for performing rapid microinjection followed by CLEM (Fig. 1). The microinjection CLEM procedure can be used to introduce specific quantities of small molecules and/or proteins directly into the eukaryotic cell cytoplasm and study the effects from millimeter to multi-nanometer resolution (Fig. 2). The technique is based on microinjecting cells grown on laser etched glass gridded coverslips affixed to the bottom of live cell dishes and imaging with both confocal fluorescent and electron microscopy. Localization of the cell(s) of interest is facilitated by the grid pattern, which is easily transferred, along with the cells of interest, to the Epon resin used for immobilization of samples and sectioning prior to electron microscopy analysis (Fig. 3). Overlay of fluorescent and EM images allows the user to determine the subcellular localization as well as any morphological and/or ultrastructural changes induced by the microinjected molecule of interest (Fig. 4). This technique is amenable to time points ranging from &le;5 s up to several hours, depending on the nature of the microinjected sample.

Correlative Light and Electron Microscopy CLEM as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The CLEM technique has been adapted to analyze ultrastructural morphology of membranes, organelles, and subcellular structures affected by microinjected molecules. This method combines the powerful techniques of micromanipulation/microinjection, confocal fluorescent microscopy, and electron microscopy to allow millimeter to multi-nanometer resolution. This technique is amenable to a wide variety of applications.


The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity ...

Establishment of Microbial Eukaryotic Enrichment Cultures from a Chemically Stratified Antarctic Lake and Assessment of Carbon Fixation Potential

Lake Bonney is one of numerous permanently ice-covered lakes located in the McMurdo Dry Valleys, Antarctica. The perennial ice cover maintains a chemically stratified water column and unlike other inland bodies of water, largely prevents external input of carbon and nutrients from streams. Biota are exposed to numerous environmental stresses, including year-round severe nutrient deficiency, low temperatures, extreme shade, hypersalinity, and 24-hour darkness during the winter 1. These extreme environmental conditions limit the biota in Lake Bonney almost exclusively to microorganisms 2.
Single-celled microbial eukaryotes (called "protists") are important players in global biogeochemical cycling 3 and play important ecological roles in the cycling of carbon in the dry valley lakes, occupying both primary and tertiary roles in the aquatic food web. In the dry valley aquatic food web, protists that fix inorganic carbon (autotrophy) are the major producers of organic carbon for organotrophic organisms 4, 2. Phagotrophic or heterotrophic protists capable of ingesting bacteria and smaller protists act as the top predators in the food web 5. Last, an unknown proportion of the protist population is capable of combined mixotrophic metabolism 6, 7. Mixotrophy in protists involves the ability to combine photosynthetic capability with phagotrophic ingestion of prey microorganisms. This form of mixotrophy differs from mixotrophic metabolism in bacterial species, which generally involves uptake dissolved carbon molecules. There are currently very few protist isolates from permanently ice-capped polar lakes, and studies of protist diversity and ecology in this extreme environment have been limited 8, 4, 9, 10, 5. A better understanding of protist metabolic versatility in the simple dry valley lake food web will aid in the development of models for the role of protists in the global carbon cycle.
We employed an enrichment culture approach to isolate potentially phototrophic and mixotrophic protists from Lake Bonney. Sampling depths in the water column were chosen based on the location of primary production maxima and protist phylogenetic diversity 4, 11, as well as variability in major abiotic factors affecting protist trophic modes: shallow sampling depths are limited for major nutrients, while deeper sampling depths are limited by light availability. In addition, lake water samples were supplemented with multiple types of growth media to promote the growth of a variety of phototrophic organisms.
RubisCO catalyzes the rate limiting step in the Calvin Benson Bassham (CBB) cycle, the major pathway by which autotrophic organisms fix inorganic carbon and provide organic carbon for higher trophic levels in aquatic and terrestrial food webs 12. In this study, we applied a radioisotope assay modified for filtered samples 13 to monitor maximum carboxylase activity as a proxy for carbon fixation potential and metabolic versatility in the Lake Bonney enrichment cultures.

Microbial eukaryotes are both a source of photosynthetically-derived carbon and top predatory species in permanently ice-covered Antarctic lakes. This report describes an enrichment culture approach to isolate metabolically versatile microbial eukaryotes from the Antarctic lake, Lake Bonney, and assesses inorganic carbon fixation potential using a radioisotope assay for Ribulose-1,5-bisphophate carboxylase oxygenase (RubisCO) activity.

Lake Bonney is one of numerous permanently ice-covered lakes located in the McMurdo Dry Valleys, Antarctica. The perennial ice cover maintains a ...

Metabolic Profile Analysis of Zebrafish Embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and composed of mature enterocytes, goblet cells and enteroendocrine cells; and crypts, residing proximal to the submucosa and the muscularis, harboring adult stem and progenitor cells and mature Paneth cells, as well as stromal and immune cells of the crypt microenvironment. Until the last few years, in vitro studies of small intestine was limited to cell lines derived from either benign or malignant tumors, and did not represent the physiology of normal intestinal epithelia and the influence of the microenvironment in which they reside. Here, we demonstrate a method adapted from Sato et al. (2009) for culturing primary mouse intestinal crypt organoids derived from C57BL/6 mice. In addition, we present the use of crypt organoid cultures to assay the crypt metabolic profile in real time by measurement of basal oxygen consumption, glycolytic rate, ATP production and respiratory capacity. Organoids maintain properties defined by their source and retain aspects of their metabolic adaptation reflected by oxygen consumption and extracellular acidification rates. Real time metabolic studies in this crypt organoid culture system are a powerful tool to study crypt organoid energy metabolism, and how it can be modulated by nutritional and pharmacological factors.

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Small intestinal crypt organoids cultured ex vivo provide a tissue culture system that recapitulates growth of crypts dependent on stem cells and their niche. We established a method to assay the metabolic profile in real time in primary mouse crypt organoids. We found organoids maintain physiological properties defined by their source.

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and ...

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors&#160;induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment&#160;was&#160;required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by &#966;29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 1010 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30,&#160;HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

This paper presents a sensitive method called Circle-Seq for purifying extrachromosomal circular DNA (eccDNA). The method encompasses column purification, removal of remaining linear chromosomal DNA, rolling-circle amplification and high-throughput sequencing. Circle-Seq is applicable to genome-scale screening of eukaryotic eccDNA and studying genome instability and copy-number variation.

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs ...

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy (SEM)

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to cells, tissues, organelles, and even whole organisms. This protocol focuses on two chemical drying methods, hexamethyldisilazane (HMDS) and t-butyl alcohol (TBA), and their application to imaging of both prokaryotic and eukaryotic organisms using SEM. In this article, we describe how to fix, wash, dehydrate, dry, mount, sputter coat, and image three types of organisms: cyanobacteria (Toxifilum mysidocida, Golenkina sp., and an unknown sp.), two euglenoids from the genus Monomorphina (M. aenigmatica and M. pseudopyrum), and the fruit fly (Drosophila melanogaster). The purpose of this protocol is to describe a fast, inexpensive, and simple method to obtain detailed information about the structure, size, and surface characteristics of specimens that can be broadly applied to a large range of organisms for morphological assessment. Successful completion of this protocol will allow others to use SEM to visualize samples by applying these techniques to their system.

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy SEM

SEM analysis is an effective method to aid in species identification or phenotypic discrimination. This protocol describes the methods for examining specific morphological details of three representative types of organisms and would be broadly applicable to examining features of many organismal and tissue types.

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to ...