We would like to thank Dr. Paul Frost for use of his microrespirometer, and Nicole Wagner and Alex Bell for technical assistance. This work was funded by an NSERC grant to B.J.S.

1. Corn Cob Infection
<ol>
	<li>Grow Zea mays (cv. Golden Bantam) until cobs are formed and have started to silk (approximately 60 days).</li>
	<li>Culture compatible haploid U. maydis strains using standard protocols as previously described7.</li>
	<li>Infect corn cobs using standard protocols as previously described7.</li>
</ol>
2. Teliospore Harvesting
<ol>
	<li>Autoclave equipment (B&#252;chner funnels, B&#252;chner flasks, blenders, 250 mL centrifuge bottles, flat spatulas, and water) using a standard dry cycle with at least 30 min sterilization at 121 &#176;C (standard liquid cycle for water).</li>
	<li>Remove infected cobs from plants (approximately 28&#8211;35 days post infection) using a razor and set the cobs on a tray covered in bench protector.</li>
	<li>Remove the tumours from cobs with a razor blade and collect in a beaker.</li>
	<li>Fill a 250 mL laboratory blender cup with tumours until approximately 1/3 full and add autoclaved dH2O until the blender cup is approximately 3/4 full. Disrupt the tumours by pulsing the blender at low, until homogenized.</li>
	<li>Connect the vacuum pump to a water trap and then to a 1 L B&#252;chner flask.</li>
	<li>Insert large B&#252;chner funnel into the flask and line the bottom of the B&#252;chner Funnel with four layers of cheesecloth.</li>
	<li>Turn on the vacuum pump.</li>
	<li>Pour a portion of the homogenized tumours through the cheesecloth and scrape with a spatula.</li>
	<li>Pour some dH2O into the cheesecloth in order to flush the teliospores through.</li>
	<li>Repeat until the dH2O coming through the cheesecloth is clear.</li>
	<li>Wring out the cheesecloth containing the homogenized tumour material into the filter to ensure maximum teliospore recovery.</li>
	<li>When the 1 L B&#252;chner flask is getting close to full, empty it into a large Erlenmeyer flask and set it aside.</li>
	<li>Put a new piece of cheesecloth into the filter and repeat the steps (2.7&#8211;2.12) until all of the tumours have been disrupted and filtered.</li>
	<li>Pour the filtered teliospores into autoclaved 250 mL centrifuge bottles and centrifuge at 1,000 x g for 5 min, and decant the supernatant.</li>
	<li>Repeat step 2.14 until all of the filtered teliospores are pelleted by centrifugation.</li>
	<li>Suspend the pellets in a small amount of water and transfer to 50 mL centrifuge tubes.</li>
	<li>Centrifuge the tubes at 1,000 x g for 5 min, and decant the supernatant.</li>
	<li>Suspend the pellet in approximately 50 mL of dH2O, centrifuge the tubes at 1,000 x g for 5 min, and decant the supernatant. Gently scrape off the gray top layer with a spatula and dispose of it. Repeat until there is no longer a gray layer on top.</li>
	<li>Dry the samples overnight in a vacuum desiccator.</li>
	<li>Store dried teliospores at 4 &#176;C until use.</li>
	<li>If desired, treat the teliospores with copper sulphate8 before inducing to germinate. If not treated, then perform thorough microscopic analysis of the teliospores to confirm the sample represents pure teliospores and is devoid of bacterial or other contaminations.
	<ol>
		<li>Weigh out approximately 50 mg of teliospores in a 1.5 mL microcentrifuge tube.</li>
		<li>Add approximately 1.0 mL of 0.75% CuSO4 to the 1.5 mL microcentrifuge tube containing the teliospores. Pipette up and down to suspend the teliospores in the CuSO4 solution followed by agitating the sample for 3 h.</li>
		<li>Centrifuge the sample at 2,500 x g for 5 min and remove the supernatant. Resuspend the teliospore pellet with sterile water, repeat the centrifugation, and remove the supernatant.</li>
		<li>Repeat step 2.21.3 two more times.</li>
	</ol>
	</li>
</ol>
3. Teliospore Viability and Germination Test
<ol>
	<li>Weigh out approximately 10 mg of U. maydis teliospores in a 1.5 mL microcentrifuge tube to assess their viability and the timing of germination.</li>
	<li>In a biosafety cabinet, prepare potato dextrose broth (PDB, 24 g/L) supplemented with streptomycin sulfate (160 &#181;g/mL).</li>
	<li>Suspend the teliospores in 500 &#181;L of PDB. Gently pipette to mix and break up all clumps of teliospores.</li>
	<li>Transfer the teliospore suspension to an autoclaved 250 mL Erlenmeyer flask containing 10 mL of the PDB.</li>
	<li>Incubate the flask at 28 &#176;C shaking at 90 rpm for 12&#8211;16 h.</li>
	<li>In a biosafety cabinet, remove a 20 &#181;L sample of the teliospores induced to germinate and prepare a microscope slide.</li>
	<li>Using a microscope, visually assess stages of germination that are present and the presence of bacterial contamination.
	<ol>
		<li>Count the number of teliospores at stages I through V using a hemocytometer and determine the percent that have germinated.</li>
		<li>If only stage I teliospores are present, continue to incubate the flask for a total of 24 h before assessing teliospore germination. Continue incubation for a maximum of 48 h before deeming the sample non-viable.</li>
		<li>If bacterial contamination is present, supplement the PDB with kanamycin sulfate (50 &#181;g/mL) as well as streptomycin sulfate (160 &#181;g/mL) and then repeat steps 3.1 to 3.7. If bacterial contamination persists, treat teliospores with copper sulfate and repeat steps 3.1 to 3.7.</li>
	</ol>
	</li>
</ol>
4. Induction of Germination for Respiration Monitoring
<ol>
	<li>Weigh equal amount (e.g., 50 mg) of teliospores for each respiration experiment.</li>
	<li>In a biosafety cabinet, add teliospores to an autoclaved respiration chamber.</li>
	<li>Fill the chamber with PDB (24 g/L) supplemented with streptomycin sulfate (160 &#181;g/mL) and kanamycin sulfate (50 &#181;g/mL).</li>
	<li>Pipette up and down to create a teliospore suspension.</li>
	<li>Place the chamber lid in the chamber to create air tight seal.</li>
</ol>
5. Obtaining Oxygen Consumption Rate (OCR) Measurements
<ol>
	<li>Place the chamber in the chamber rack inside a water bath (preheated to 28 &#176;C).</li>
	<li>Place the O2 probe inside the opening of the chamber.</li>
	<li>Monitor the data points appearing in real time on the &#34;SensorTrace Rate&#34; program, and let the probe stabilize (~3 min after the probe is placed in the chamber).</li>
	<li>Click &#34;Measure&#34; to measure O2 levels continuously for 6 h with measurements recorded at 2 s intervals.</li>
	<li>Stop the measurement, and repeat steps 4.1&#8211;5.4 for each sample to be analyzed.</li>
	<li>Export the data to Microsoft Excel by clicking &#34;File | Export | Save as .xls&#34;.</li>
</ol>
6. Data Analysis
<ol>
	<li>Calculate OCR
	<ol>
		<li>In the exported Excel file under the &#34;Within_Rates&#34; tab, record the &#34;Rate&#34; for each chamber measurement (nmol/h).</li>
		<li>For each experimental sample, subtract the &#34;Rate&#34; of the blank chamber from the &#34;Rate&#34; of the experimental sample chamber to obtain a corrected OCR value, and take the absolute value of this number.</li>
		<li>Calculate the total OCR per mg of teliospores by dividing the corrected absolute OCR value by the cellular mass used.</li>
		<li>Average replicate &#34;OCR per mg of teliospores&#34; values for each strain.</li>
	</ol>
	</li>
	<li>Analyze the data using appropriate statistical method (e.g., student&#39;s t-test, analysis of variance) using Microsoft Excel of other statistical software.</li>
	<li>To graph raw data, calculate the percentage of oxygen remaining for each time-point you wish to graph. Divide first reading by itself and multiply by 100 (100% oxygen remaining), then divide each subsequent reading by the first reading, and multiply by 100 to obtain the percent of oxygen remaining in the chamber.</li>
</ol>
7. Induction of Teliospore Germination to Isolate Teliospores at Distinct Stages of Germination
<ol>
	<li>Prepare PDB (24 g/L) supplemented with streptomycin sulfate (160 &#181;g/mL) in a biosafety cabinet.</li>
	<li>Place approximately 10 mg of U. maydis teliospores into a 1.5 mL microcentrifuge tube.</li>
	<li>Suspend the teliospores in 500 &#181;L of PDB. Gently pipette to mix until there are no clumps of teliospores in the medium.</li>
	<li>Transfer the teliospore suspension to an autoclaved 250 mL Erlenmeyer flask containing PDB supplemented with streptomycin sulfate.</li>
	<li>Incubate the flask overnight at 28 &#176;C shaking at 90 rpm.</li>
</ol>
8. Preparation of Petri Dish and Micromanipulator
<ol>
	<li>Prepare a Petri dish (57 cm2) by pipetting rows of droplets for microcapillary preparation, and sample collection.
	<ol>
		<li>Pipette 5 &#181;L (x4) dH2O droplets across the top of the Petri dish.</li>
		<li>Pipette 2 &#181;L (x3) of RNA stabilization solution on the Petri dish to be used for sample collection.</li>
		<li>Pipette 5 &#181;L (x30) droplets of germinating teliospores on the Petri dish.</li>
	</ol>
	</li>
	<li>Add 15 mL of mineral oil to the petri dish. Ensure that all droplets are covered by oil before proceeding.</li>
	<li>Prepare a microcapillary with a 15 &#181;m inner diameter, 1 mm flange, 55 mm length, and a 20&#176; tip angle by placing it in the microcapillary holder and submerging it in the mineral oil where capillary action will allow the mineral oil to enter the microcapillary. Release the pressure in the microcapillary before bringing it to the water droplet. Aspirate to prepare the microcapillary with water.</li>
</ol>
9. Isolation of Stage-specific Germinating Teliospores
<ol>
	<li>Using the controls of the micromanipulator, move the prepared microcapillary to one of the germination droplets. Penetrate the droplet, lower the microcapillary, and bring the mouth of the microcapillary up to a germinating teliospore at the stage of germination of interest.</li>
	<li>Slowly aspirate to capture the germinating teliospore. Stop aspirating once the teliospore has entered the microcapillary. Repeat until there are approximately five teliospores in the microcapillary.</li>
	<li>Raise the microcapillary with the micromanipulator and bring it to the collection droplet of RNA stabilization solution. Penetrate the droplet and inject the teliospores into the droplet.</li>
	<li>Repeat steps 8.1 to 8.3 until approximately 1,000 teliospores have been captured.</li>
</ol>
10. Recovery of Collection Droplet
<ol>
	<li>Pipette up the collection droplet and transfer it to the lid of an RNase/DNase-free 2.0 mL microcentrifuge tube. Carefully remove the mineral oil with a pipette without disturbing the collection droplet.</li>
	<li>Use the teliospores for downstream applications such as RNA isolation.</li>
</ol>

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	<li>Saville, B. J., Donaldson, M. E., Doyle, C. E., Swan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Meiosis - Molecular Mechanisms and Cytogenetic Diversity. , 411-460 (2012).</li><li>Christensen, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Monograph Number 2. , (1963).</li><li>Caltrider, P. D., Gottlieb, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+activity+and+enzymes+for+glucose+catabolism+in+fungal+spores.">Respiratory activity and enzymes for glucose catabolism in fungal spores.</a> Phytopathology. 53, 1021-1030 (1963).</li><li>Allen, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+aspects+of+spore+germination+in+fungi.">Metabolic aspects of spore germination in fungi.</a> Ann. Rev. Phytopathol. 3, 313-342 (1965).</li><li>Warburg, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+of+tumours.">Metabolism of tumours.</a> Biochemische Zeitschrift. 142, 317-333 (1923).</li><li>Ostrowski, L. A., Saville, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+antisense+transcripts+are+linked+to+the+modulation+of+mitochondrial+function+and+teliospore+dormancy+in+Ustilago+maydis.">Natural antisense transcripts are linked to the modulation of mitochondrial function and teliospore dormancy in Ustilago maydis.</a> Mol Microbiol. 103 (5), 745-763 (2017).</li><li>Morrison, E. N., Donaldson, M. E., Saville, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+analysis+of+genes+expressed+in+the+Ustilago+maydis+dikaryon:+uncovering+a+novel+class+of+pathogenesis+genes.">Identification and analysis of genes expressed in the Ustilago maydis dikaryon: uncovering a novel class of pathogenesis genes.</a> Canadian Journal of Plant Pathology-Revue Canadienne De Phytopathologie. 34 (3), 417-435 (2012).</li><li>Doyle, C. E., Cheung, H. Y. K., Spence, K. L., Saville, B. 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The authors have no competing financial interests or other conflicts of interest to disclose.

Basidiomycete biotrophic plant pathogens cause billions of dollars in crop losses annually. The vast majority of these pathogens produce teliospores that are integral to fungal dispersal and sexual reproduction. Gaining knowledge of the development and germination of teliospores is critical to understanding the spread of the devastating diseases caused by these fungi. In order to identify molecular changes at key control points we have devised a method to identify the timing of physiological shifts and another to isolate...

The smut fungi (Ustilaginales) consist of over 1,600 species that infect grasses including the important cereal crops of corn, barley, and wheat, causing billions of dollars in crop losses annually1. These fungi are characterized by the production of teliospores, which have darkly pigmented cell walls and are the dispersal agents. Teliospores function to shield genetic material during the stresses of dispersal between host plants, and can persist in a dormant state for years2. As such, teliospores are an essential component of disease spread.
In order to study teliospore biology, our laboratory utilizes the model smut fungus Ustilago maydis (U. maydis), which is the causal agent of the disease &#39;common smut of corn&#39;. Mature U. maydis teliospores are characterized by growth arrest, reduced cellular metabolism, and low levels of cellular respiration3. In favorable environmental conditions (e.g., the presence of specific sugars), U. maydis teliospores germinate and complete meiosis, producing basidiospores which can initiate new rounds of infection. Germination is characterized by increased respiration, the return to metabolic activity, and the progression through observable morphological stages of germination4.
The initial stage of germination includes increased respiration and metabolic function, however, there are no morphological indications of change. The original measurements of respiratory change in U. maydis were carried out over 50 years ago, measuring oxygen consumption manometrically with a Warburg flask apparatus5. We have developed a new, simple method of studying precise changes in respiration during teliospore germination by measuring oxygen consumption over a time course of germination using a Clark-type microrespirometer. We previously used this method to study changes in respiratory rate between wild-type U. maydis haploid cells and mutants with defective mitochondria6, and have adapted the protocol here to study changes in teliospore respiration during germination. This provides a means of accurately identifying the timing of respiration change so that we can target teliospores at the appropriate time after the initiation of germination to investigate early molecular events. The progression of germination can be followed microscopically once the promycelia emerges from the teliospore, but the asynchronous nature inhibited the isolation of enough teliospores at a given stage for investigation. We developed a microdissection technique similar to those used for in vitro fertilization to physically collect teliospores at distinct morphological stages of germination.

<table border="0" cellpadding="0" cellspacing="0" style="width:707px;" width="707">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:437px;">Streptomycin Sulfate</td>
			<td style="width:75px;">BioShop</td>
			<td style="width:124px;">STP101</td>
			<td style="width:71px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Kanamycin Sulfate</td>
			<td>BioShop</td>
			<td>KAN201</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potato Dextrose Broth</td>
			<td>BD Difco</td>
			<td>254920</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 L Waring Laboratory blender</td>
			<td>Waring</td>
			<td>7011S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cheesecloth</td>
			<td>VWR</td>
			<td>470150-438</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nalgene Polypropylene Desiccator with Stopcock</td>
			<td>ThermoFisher Scientific</td>
			<td>5310-0250</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Unisense MicroRespiration system</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">MicroRespiration Sensor (O2)</td>
			<td>Unisense</td>
			<td>OX10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MicroOptode Meter Amplifier</td>
			<td>Unisense</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MR-Ch Small</td>
			<td>Unisense</td>
			<td>MR-Ch</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SensorTrace Rate Software</td>
			<td>Unisense</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MicroRespiration Rack</td>
			<td>Unisense</td>
			<td>MR2-Rack</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MicroRespiration Stirrer</td>
			<td>Unisense</td>
			<td>MR2-Co</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microdissection system</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Axio Vert.A1 Inverted Light Microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Coarse Manipulator</td>
			<td>Narishige</td>
			<td>MMN-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Three-axis Hanging Joystick Oil Hydraulic Micromanipulator</td>
			<td>Narishige</td>
			<td>MMO-202ND</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pneumatic Microinjector</td>
			<td>Narishige</td>
			<td>IM-11-2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TransferTip (ES)</td>
			<td>Eppendorf</td>
			<td>5175107004</td>
			<td></td>
		</tr>
	</tbody>
</table>

investigating teliospore germination using microrespiration analysis and microdissection

Smut fungi are the etiological agents of several devastating agricultural diseases. They are characterized by the production of teliospores, which are thick-walled dispersal agents. Teliospores can remain dormant for decades. The dormancy is characterized by low metabolic rates, paused macromolecular biosynthesis and greatly reduced levels of respiration. Upon receiving required environmental signals, teliospores germinate to produce haploid cells, which can initiate new rounds of infection. Teliospore germination is characterized by the resumption of macromolecular biosynthesis, increased respiration and dramatic morphological changes. In order to precisely measure changes in cellular respiration during the early stages of germination, we have developed a simple protocol employing a Clark-type respirometer. The later stages of germination are distinguished by specific morphological changes, but germination is asynchronous. We developed a microdissection technique that enables us to collect teliospores at distinct germination stages.

Using the Clark-type microrespirometer-based method of measuring changes in respiration during teliospore dormancy and germination, we confirmed that dormant teliospores exhibit a low level of respiration (~1,075 &#181;mol/h/mg) compared to germinating teliospores (~2,614 &#181;mol/h/mg; Figure 1A). This represents a ~2.4-fold change in average rate of respiration between dormant teliospores and teliospores that have been induced to germinate. In addition, we...

Watch this Scientific Journal Video about Investigating Teliospore Germination Using Microrespiration Analysis and Microdissection at JoVE.com

Investigating Teliospore Germination Using Microrespiration Analysis and Microdissection

Smut fungi cause many devastating agricultural diseases. They are dispersed as dormant teliospores that germinate in response to environmental cues. We outline two methods to investigate molecular changes during germination: measuring respiration increase to detect metabolic activation and assessing changing molecular events by isolating teliospores at distinct morphological stages.

Smut fungi are the etiological agents of several devastating agricultural diseases. They are characterized by the production of teliospores, which are ...

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7.2K Views.  Trent University. Smut fungi cause many devastating agricultural diseases. They are dispersed as dormant teliospores that germinate in response to environmental cues. We outline two methods to investigate molecular changes during germination: measuring respiration increase to detect metabolic activation and assessing changing molecular events by isolating teliospores at distinct morphological stages.

Video: Investigating Teliospore Germination Using Microrespiration Analysis and Microdissection

Laser Capture Microdissection of Mammalian Tissue

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or formalin-fixed, paraffin-embedded tissue sections. LMD techniques rely on a thermo labile membrane placed either on top of, or underneath, the tissue section. In one method, focused laser energy is used to melt the membrane onto the underlying cells, which can then be lifted out of the tissue section. In the other, the laser energy vaporizes the foil along a path "drawn" on the tissue, allowing the selected cells to fall into a collection device. Each technique allows the selection of cells with a minimum resolution of several microns. DNA, RNA, protein, and lipid samples may be isolated and analyzed from micro-dissected samples. In this video, we demonstrate the use of the Leica AS-LMD laser microdissection instrument in seven segments, including an introduction to the principles of LMD, initializing the instrument for use, general considerations for sample preparation, mounting the specimen and setting up capture tubes, aligning the microscope, adjusting the capture controls, and capturing tissue specimens. Laser-capture micro-dissection enables the investigator to isolate samples of pure cell populations as small as a few cell-equivalents. This allows the analysis of cells of interest that are free of neighboring contaminants, which may confound experimental results.

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or ...

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 ...

Microdissection of Zebrafish Embryonic Eye Tissues

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post fertilization (dpf), the larvae will show the first visual response. Many genes have been identified to control a proper eye development, but we are far from a complete understanding of the underlying genetic architecture. Whole genome gene expression profiling is a useful tool to elucidate genetic regulatory network for eye development. However, the small size of the embryonic eye in zebrafish makes it challenging to obtain intact and pure eye tissues for expression analysis. For example, the anterior-posterior length of the eye between day 2 and 3 is only approximately 200-300 &mu;m, while the diameter of the lens is less 100 &mu;m. Also, the retinal pigment epithelium (RPE) underlying the retina is just a single-layer epithelium. While gene expression profiles can be obtained from the whole embryo, they do not accurately represent the expression of these tissues. Therefore pure tissue must be obtained for a successful gene expression profiling of eye development. To address this issue, we have developed an approach to microdissect intact retina and retina with RPE attached from 1-3 dpf, which cover major stages of eye morphogenesis. All procedures can be done with fine forceps and general laboratory supplies under standard stereomicroscopes. For retinal dissection, the single-layer RPE is removed and peeled off by brushing action and the preferential adherence of the RPE remnants to the surface of the culture plate for dissection. For RPE-attached retinal dissection, the adherence of RPE to the dissection plate is removed before the dissection so that the RPE can be completely preserved with the retina. A careful lifting action of this tissue can efficiently separate the presumptive choroid and sclera. The lens can be removed in both cases by a chemically etched tungsten needle. In short, our approach can obtain intact eye tissues and has been successfully utilized to study tissue-specific expression profiles of zebrafish retina1, 2 and retinal pigment epithelium3.

This article describes an approach to microdissect zebrafish retinas with and without retinal pigment epithelium attached, from one to three days postfertilization embryos.

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post ...

Fruit Volatile Analysis Using an Electronic Nose

Numerous and diverse physiological changes occur during fruit ripening, including the development of a specific volatile blend that characterizes fruit aroma. Maturity at harvest is one of the key factors influencing the flavor quality of fruits and vegetables1. The validation of robust methods that rapidly assess fruit maturity and aroma quality would allow improved management of advanced breeding programs, production practices and postharvest handling. 
Over the last three decades, much research has been conducted to develop so-called electronic noses, which are devices able to rapidly detect odors and flavors2-4. Currently there are several commercially available electronic noses able to perform volatile analysis, based on different technologies. The electronic nose used in our work (zNose, EST, Newbury Park, CA, USA), consists of ultra-fast gas chromatography coupled with a surface acoustic wave sensor (UFGC-SAW). This technology has already been tested for its ability to monitor quality of various commodities, including detection of deterioration in apple5; ripeness and rot evaluation in mango6; aroma profiling of thymus species7; C6 volatile compounds in grape berries8; characterization of vegetable oil9 and detection of adulterants in virgin coconut oil10. 
This system can perform the three major steps of aroma analysis: headspace sampling, separation of volatile compounds, and detection. In about one minute, the output, a chromatogram, is produced and, after a purging cycle, the instrument is ready for further analysis. The results obtained with the zNose can be compared to those of other gas-chromatographic systems by calculation of Kovats Indices (KI). Once the instrument has been tuned with an alkane standard solution, the retention times are automatically converted into KIs. However, slight changes in temperature and flow rate are expected to occur over time, causing retention times to drift. Also, depending on the polarity of the column stationary phase, the reproducibility of KI calculations can vary by several index units11. A series of programs and graphical interfaces were therefore developed to compare calculated KIs among samples in a semi-automated fashion. These programs reduce the time required for chromatogram analysis of large data sets and minimize the potential for misinterpretation of the data when chromatograms are not perfectly aligned.
We present a method for rapid volatile compound analysis in fruit. Sample preparation, data acquisition and handling procedures are also discussed.

A rapid method for volatile compound analysis in fruit is described. The volatile compounds present in the headspace of a homogenate of the sample are rapidly separated and detected with ultra-fast gas chromatography (GC) coupled with a surface acoustic wave (SAW) sensor. A procedure for data handling and analysis is also discussed.

Numerous and diverse physiological changes occur during fruit ripening, including the development of a specific volatile blend that characterizes fruit ...

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for the conversion and replication of their benign cellular isoform. Accumulating evidence suggests that many protein aggregates can act as self-propagating templates and corrupt the folding of cognate proteins. Although aggregates can be functional under certain circumstances, this process often leads to the disruption of the cellular protein homeostasis (proteostasis), eventually leading to devastating diseases such as Alzheimer&#8217;s disease (AD), Parkinson&#8217;s disease (PD), Amyotrophic lateral sclerosis (ALS), or transmissible spongiform encephalopathies (TSEs). The exact mechanisms of prion propagation and cell-to-cell spreading of protein aggregates are still subjects of intense investigation. To further this knowledge, recently a new metazoan model in Caenorhabditis elegans, for expression of the prion domain of the cytosolic yeast prion protein Sup35 has been established. This prion model offers several advantages, as it allows direct monitoring of the fluorescently tagged prion domain in living animals and ease of genetic approaches. Described here are methods to study prion-like behavior of protein aggregates and to identify modifiers of prion-induced toxicity using C. elegans.

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prion-like propagation of protein aggregates has recently emerged as being implicated in many neurodegenerative diseases. The goal of this protocol is to describe, how to use the nematode C. elegans as a model system to monitor protein spreading and to investigate prion-like phenomena.

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for ...

RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires precise identification of cells subpopulations from a heterogeneous tissue. Identification of cells of interest for LCM is usually based on morphological criteria or on fluorescent protein reporters. The combination of LCM and rapid immunolabeling offers an alternative and efficient means to visualize specific cell types and to isolate them from surrounding tissue. High-quality RNA can then be extracted from a pure cell population and further processed for downstream applications, including RNA-sequencing, microarray or qRT-PCR. This approach has been previously performed and briefly described in few publications. The goal of this article is to illustrate how to perform rapid immunolabeling of a cell population while keeping RNA integrity, and how to isolate these specific cells using LCM. Herein, we illustrated this multi-step procedure by immunolabeling and capturing dopaminergic cells in brain tissue from one-day-old mice. We highlight key critical steps that deserve special consideration. This protocol can be adapted to a variety of tissues and cells of interest. Researchers from different fields will likely benefit from the demonstration of this approach.

Gene expression analysis of a subset of cells in a specific tissue represents a major challenge. This article describes how to isolate high-quality total RNA from a specific cell population by combining a rapid immunolabeling method with laser capture microdissection.

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires ...

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The colonic epithelium is dynamically turned over and epithelial cells are generated in the stem cell containing crypts of Lieberk&#252;hn. Progenitor cells produced in the crypt-bases migrate toward the luminal surface, undergoing a process of cellular differentiation before being shed into the gut lumen. In order to study these processes at the molecular level, we have developed a simple method for the microdissection of two spatially distinct regions of the colonic mucosa; the proliferative crypt zone, and the differentiated surface epithelial cells. Our objective is to isolate specific crypt and surface epithelial cell populations from mouse colonic mucosa for the isolation of RNA and protein.

Here we describe a simple method for the isolation of spatially distinct murine colonic epithelial surface cells from the underlying crypt-base cells.

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The ...

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 ...