Name: comprehensive compositional analysis of plant cell walls (lignocellulosic biomass) part ii: carbohydrates
Uploaded: 2010-03-12T17:00:00
Duration: 10 min 46 s
Description: Watch this Scientific Journal Video about Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part II: Carbohydrates at JoVE.com

We are grateful to Matthew Robert Weatherhead for excellent technical service and John Ralph, University of Wisconsin for valuable advice, discussions, and the poplar wood sample. This work was funded by the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Science, U.S. Department of Energy (award no. DE-FG02-91ER20021) and by the US Department of Energy (DOE) Great Lakes Bioenergy Research Center (DOE BER Office of Science DE-FC02-07ER64494).

1. Cell wall isolation
<ol>
	<li>grind roughly 60-70mg of air- or freeze dried plant material with 5.5 mm stainless steel balls in a 2ml sarstedt screw cap tube using a retschmill (1 min, 25 Hz). An alternative, the use of a high-throughput grinding and dispensing robot termed iWall is described in Part I3.</li>
<li>remove the steel balls before continuing with the cell wall isolation procedure 
The detailed protocol of the preparation of cell wall material is shown in Part I3. For completeness here the written steps of the protocol. </li>
<li>add 1.5 ml of 70% aqueous ethanol, and vortex thoroughly</li>
<li>centrifuge at 10,000 rpm for 10 min to pellet the alcohol insoluble residue</li>
<li>aspirate or decant the supernatant</li>
<li>add 1.5 ml of chloroform/methanol (1:1 v/v) solution to the residue and shake tube thoroughly to resuspend the pellet</li>
<li>centrifuge at 10,000 rpm for 10 min and aspirate or decant the supernatant</li>
<li>resuspend pellet in 500 ul of acetone </li>
<li>evaporate the solvent with a stream of air at 35&deg;C until dry 
 If needed dried samples can be stored at room-temperature until further processing.</li>
<li>To initiate the removal of starch from the sample re-suspend the pellet in 1.5 ml of a 0.1 M sodiumacetate buffer pH 5.0.</li>
<li>cap the sarstedt tubes and heat for 20 min. at 80&deg;C in a heating block.</li>
<li>cool the suspension on ice</li>
<li>add the following agents to the pellet: 35 &mu;l of 0.01% Sodiumazide (NaN3), 35 &mu;l Amylase (50 &mu;g/1mL H2O; from Bacillus species, SIGMA); 17 &mu;l Pullulanase (18.7 units from bacillus acidopullulyticus; SIGMA). Cap the tube and vortex thoroughly. </li>
<li>The suspension is incubated over night at 37&deg;C in the shaker. Orienting the tubes horizontally aides improved mixing. </li>
<li>heat suspension at 100&deg;C for 10 min in a heating block to terminate digestion.</li>
<li>centrifuge (10,000 rpm, 10 min) and discard supernatant containing solubilized starch</li>
<li>wash the remaining pellet three times by adding 1.5 ml water, vortexing, centriguation, and decanting of the washing water.</li>
<li>resuspend pellet in 500 ul of acetone </li>
<li>evaporate the solvent with a stream of air at 35&deg;C until dry. It may be necessary also to break up the material in the tube with a spatula for better drying. 
 The dried material presents isolated cell wall (lignocellulosics). If needed dried samples can be stored at room-temperature until further processing.</li>
</ol>
2. Matrix Polysaccharide composition
This method is essentially a modification of the method published by Albersheim 1.
<ol>
 <li>To determine the monosaccharide composition of the wall material weigh 2 mg of cell wall material into 2ml starstedt tubes either by hand or in a highthroughput fashion using the iWall, a robotic grinding and weighing robot.</li>
	<li>Add 20 uL of an Inositol solution (5mg/ml) as an internal standard. For a 2 mg cell wall sample we recommend to add 100 ug.</li>
	<li>rinse tube walls with 250 ul of acetone to collect the cell wall material on the bottom of the tube, and evaporate the acetone very gentle under airflow. </li>
	<li>For the weak acid hydrolysis add 250 ul of 2M trifluoroacetic acid (TFA) to each sample. Add TFA carefully to ensure no material is splashed up onto the tube walls.</li>
	<li>cap tightly and incubate for 90 min at 121&deg;C in a heating block.</li>
	<li>cool the heating blocks and samples on ice.</li>
	<li>centrifuge the tubes at 10,000 rpm for 10 min.</li>
	<li>transfer 100 ul of the acidic supernatant containing the matrix polysaccharide derived monosaccharide to a glass screw cap vials making sure not to disturb the pellet material. The pellet can be used for the crystalline cellulose assay below (see 3.)</li>
	<li>evaporate the TFA in the glass tube under a gentle stream of air in an evaporation device. </li>
	<li>add 300 &mu;l 2-Propanol, vortex and evaporate at 25&deg;C (repeat for a total of three times)</li>
	<li>The first step of the alditol acetate derivatization procedure is to perform a reduction of the monosaccharides to their corresponding alditols. For this purpose add 200 &mu;l of a sodium borohydride solution to each dried sample. Prepare a fresh solution each time using 10mg of Sodium borohydride per 1ml of 1M Ammonium hydroxide.</li>
	<li>leave glass vial at room temperature for 1.5 hours</li>
	<li>neutralize the solution by adding 150 ul of glacial acetic acid </li>
	<li>vortex and evaporate at 25&deg;C .</li>
	<li>add 250 &mu;l Acetic acid/Methanol (1:9, v/v), vortex and evaporate at 25&#730;C</li>
	<li>add 250 &mu;l Methanol, and evaporate under stream of air (repeat for a total of three times)</li>
	<li>For the acetylation of the alditols, add 50 &mu;l of Acetic anhydride and 50 &mu;l of Pyridine, vortex and incubate for 20 min at 121&deg;C in a heating block. </li>
	<li>cool samples in the block down with ice while wait for temp decrease to approximately room temperature.</li>
	<li>evaporate the reagents under a gentle stream of air at room temperature. Be careful: alditol acetates are highly volatile. </li>
	<li>add 200 &mu;l Toluene and evaporate under air (x3)</li>
	<li>In the final steps the alditol acetates are extracted. First, add 500 ul of ethyl acetate and swirl lightly.</li>
	<li>add 2 ml of water, cap tubes and vortex.</li>
	<li>centrifuge tubes at 2,000 RPM for 5 min to obtain clear separate layers (ethyl acetate on top, water on bottom)</li>
	<li>pipette 50 ul of the ethyl acetate layer into GC/MS vials with inserts.</li>
	<li>dilute by adding 100 ul of acetone to the GC-vial and cap. The sample volume and dilution volumes can be adjusted to avoid overloading the GC/MS if the sample concentration is too high. The GC-vial can be stored at 4&deg;C, if the GC/MS analysis does not immediately proceed</li>
	<li>The samples are injected into a GC that is equipped with a quadrupole MS, but a flame ionization detector is also suitable. A Supelco SP-2380 (30mm X 0.25mm x 0.25 &mu;m film thickness) column is used with a 4min solvent delay and a flow rate of 1.5ml/min. Injected samples are subjected to the following temperature program: Initial hold at 160&deg;C for 2 min; a 20&deg;C/min ramp to 200&deg;C and hold for 5 min; a 20&deg;C/min ramp to 245&deg;C and hold 12 min; spike to 270&deg;C and hold for 5 min before cooling to the initial temperature of 160&deg;C 2.26.) Peaks are identified by mass profiles and/or retention times of standards. Monosaccharides are quantified based on standard curves.</li>
</ol>
3. Crystalline Cellulose Content
This method is essentially described by Updegraf8. There are a number of starting materials for this procedure: Isolated cell wall material (see 1) or wall material that has already been treated with 2M TFA (see 2.8) either the remaining pellet immediately after the acid treatment (see 2.8) or a TFA pellet, that has been washed with 2-propanol and dried.
<ol>
 <li>Add to the TFA pellet in the screw capped glass tube 1 ml of Updegraff reagent (Acetic acid: nitric acid: water, 8:1:2 v/v). </li>
	<li>Cap tube tightly, vortex, and heat in a heating block at 100&deg;C for 30 min. As a result of this treatment only crystalline cellulose remains insoluble in the pellet.</li>
	<li>Cool samples in the block on ice to room-temperature or cooler</li>
 <li>centrifuge samples at 10,000 rpm for 15 min</li>
 <li>Discard supernatant ensuring that the pellet is not disturbed and no material from the pellet is removed. For this purpose leave approx. 150 ul of supernatant in the tube. </li>
	<li>add 1.5 ml of water, shake, centrifuge, and discard supernatant as done above</li>
	<li>repeat washing procedure 3 additional times using 1.5 ml of acetone </li>
	<li>Air dry pellet very gently with air, or let dry on bench overnight</li>
	<li>the pellet (crystalline cellulose) is now completely hydrolyzed into glucose by what is called a Saeman hydrolysis. For this purpose add 175 &mu;l 72% Sulfuric acid to the sarstedt tube</li>
	<li>incubate at room temperature for 30 min, vortex and incubate for another 15 min</li>
	<li>add 825 &mu;l water and vortex</li>
	<li>centrifuge samples at 10,000 rpm for 5 min. There might be some brown insoluble material, lignin, remaining in the tube. </li>
	<li>The glucose content of the supernatant is assayed using the colorimetric anthrone assay. This assay is performed in a 96 well polystyrene microtiter plate.</li>
	<li>For the standard curve use a 1mg/ml glucose stock (stored at 0&deg;C) and create duplicate 0, 2, 4, 6, 8, and 10 ug standards by pipetting 0, 2, 4, 6, 8, and 10 ul into separate appropriate well. Fill each well up to 100 ul with water.</li>
	<li>add 10 &mu;l of each sample supernatant and 90 ml of water into separate cells but on the same microtiter plate as the standard. </li>
	<li>add 200 &mu;l of freshly prepared Anthrone Reagent (Anthrone dissolved in concentrated sulphuric acid , 2 mg anthrone/ ml sulphuric acid)</li>
	<li>heat plate for 30 min at 80&deg;C in an oven (aluminum heat spreader). Glucose containing samples turn from yellow into blue-green.</li>
	<li>let the plate cool to room-temperature and shake thoroughly. </li>
	<li>read absorption of plate at 625 mm using a microtiter plate reader.</li>
	<li>Glucose (and hence crystalline cellulose content) is calculated based on the absorbance compared to the standard curve established on the same plate.</li>
</ol>
4. Representative Results
An example of a wall analysis is presented in Figure 2. In this case poplar stem (wood) was analyzed by the various procedures outlined in the protocol section. The matrix polysaccharide composition is highlighted by an example chromatogram identifying the typical sugar present in plant cell walls, fucose, rhamnose, xylose, arabinose, galactose, mannose and glucose (and the internal standard inositol). The main hemicellulosic component of poplar is xylan as demonstrated by the high xylose content. However, the abundance of these sugars will vary depending on the feedstock used4. The glucose in this analysis is derived from the hemicellulose xyloglucan and amorphous cellulose. Due to the analysis the data can be presented as mol% or ug/ mg wall material (or dry weight). The content of crystalline cellulose is self-explanatory, one can expect values of between 20-50% of the wall dry weight. Based on the results presented here and in Part I3 the lignocellulosic composition of poplar wood is 21% lignin, 30% hemicelluloses, and 41% crystalline cellulose. The remainder would be ash.
<img src="/files/ftp_upload/1837/1837fig1.jpg" alt="Figure 1" /> Figure 1: Overview of lignocellulosic analysis. Cell walls (lignocellulosics) are isolated from crude dried plant material. The wall material is then weighted into aliquots and subdivided for the various assays. Matrix polysaccharide composition is established after treating the wall material with a weak acid (2M TFA), derivatizing the resulting solubilized monosaccharides to their alditol acetates, and analysis by GC-MS. The residue of the weak acid treatment is washed with the so-called Updegraff reagent leaving only insoluble crystlline cellulose behind. The cellulose is solubilized by sulfuric acid and quantified by a colorimetric assay determining the glucose content. In parallel, the content and composition of lignin can be determined as described in Part I3.
<img src="/files/ftp_upload/1837/1837fig2.jpg" alt="Figure 2" /> Figure 2: Comprehensive lignocellulosic analysis of poplar wood. Wood chips from poplar (Populus tremoloides) were subjected to the described protocol. Upper left: Matrix polysaccharide composition; Fuc fucose; Rha rhamnose; Ara arabinose; Xyl xylose; Man mannose; Gal galactose; Glc glucose; inositol internal standard.

<ol>
	<li>Albersheim, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+analysis+of+sugars+in+plant+cell+wall+polysaccharides+by+gas-liquid+chromatography.">A method for the analysis of sugars in plant cell wall polysaccharides by gas-liquid chromatography.</a> Carbohydr. Res. 5, 340-340 (1967).</li><li>Carroll, A., Somerville, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellulosic+Biofuels.">Cellulosic Biofuels.</a> Annu Rev Plant Biol. 60, 165-165 (2009).</li><li>Foster, C. E., Martin, T., Pauly, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+compositional+analysis+of+Plant+Cell+Walls+(Lignocellulosic+biomass),+Part+I:+Lignin.">Comprehensive compositional analysis of Plant Cell Walls (Lignocellulosic biomass), Part I: Lignin.</a> Journal of Visualized Experiments. , (2010).</li><li>Pauly, M., Keegstra, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-wall+carbohydrates+and+their+modification+as+a+resource+for+biofuels.">Cell-wall carbohydrates and their modification as a resource for biofuels.</a> Plant J. 54 (4), 559-559 (2008).</li><li>Somerville, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+a+systems+approach+to+understanding+plant-cell+walls.">Toward a systems approach to understanding plant-cell walls.</a> Science. 306 (5705), 2206-2206 (2004).</li><li>Teeri, T. T., Brumer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery,+characterisation+and+applications+of+enzymes+from+the+wood-forming+tissues+of+poplar:+Glycosyl+transferases+and+xyloglucan+endotransglycosylases.">Discovery, characterisation and applications of enzymes from the wood-forming tissues of poplar: Glycosyl transferases and xyloglucan endotransglycosylases.</a> Biocatalysis and Biotransformation. 21, 173-173 (2003).</li><li>UPDEGRAF, 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=SEMIMICRO+DETERMINATION+OF+CELLULOSE+IN+BIOLOGICAL+MATERIALS.">SEMIMICRO DETERMINATION OF CELLULOSE IN BIOLOGICAL MATERIALS.</a> Anal. Biochem. 32 (3), 420-420 (1969).</li></ol>

The described methods enable a rapid quantitative assessment of the composition of lignocellulosic plant biomass. The method allows the determination of the composition of such materials including the sugar composition of the matrix polysaccharides namely the hemicelluloses, the crystalline cellulose content. The throughput of the various analytical methods per person varies. Using the protocols described here, 20 samples can be processed for matrix polysaccharide compositions and 30 for crystalline cellulose content. Due to the quantitative nature of the data optimal feedstock crops, variety or genotypes can be assessed in terms of their suitability for biofuel production.

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		<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>Trifluoroacetic acid</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>T6508</td>
<td>&nbsp;</td>
<tr>
<td>myo-Inositol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>I5125</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Borohydride</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>213462</td>
<td>&nbsp;</td>
<tr>
<td>Pyridine</td>
<td>&nbsp;</td>
<td>J.T. Baker</td>
<td>3348-01</td>
<td>&nbsp;</td>
<tr>
<td>Acetic Anhydride</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>320102</td>
<td>&nbsp;</td>
<tr>
<td>Spectromax Plus 384</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>Plus384</td>
<td>&nbsp;</td>
<tr>
<td>GC-MS</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>7890A GC/5975C MSD</td>
<td>&nbsp;</td>
<tr>
<td>5.5mm Stainless Steel Balls</td>
<td>&nbsp;</td>
<td>Salem Ball Company</td>
<td>(N/A)</td>
<td>&nbsp;</td>
<tr>
<td>96 well plate heat spreader</td>
<td>&nbsp;</td>
<td>Biocision</td>
<td>Coolsink 96F</td>
<td>&nbsp;</td>
<tr>
<td>Retsch Mill</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>TissueLyser II</td>
<td>&nbsp;</td>
<tr>
<td>Heating block</td>
<td>&nbsp;</td>
<td>Techne</td>
<td>Dri-block DB-3D</td>
<td>&nbsp;</td>
<tr>
<td>Sample concentrator</td>
<td>&nbsp;</td>
<td>Techne</td>
<td>FSC400D</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

comprehensive compositional analysis of plant cell walls (lignocellulosic biomass) part ii: carbohydrates

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st century. This has rekindled interest in the use of plant products as industrial raw materials for the production of liquid fuels for transportation2 and other products such as biocomposite materials6. Plant biomass remains one of the greatest untapped reserves on the planet4. It is mostly comprised of cell walls that are composed of energy rich polymers including cellulose, various hemicelluloses, and the polyphenol lignin5 and thus sometimes termed lignocellulosics. However, plant cell walls have evolved to be recalcitrant to degradation as walls contribute extensively to the strength and structural integrity of the entire plant. Despite its necessary rigidity, the cell wall is a highly dynamic entity that is metabolically active and plays crucial roles in numerous cell activities such as plant growth and differentiation5. Due to the various functions of walls, there is an immense structural diversity within the walls of different plant species and cell types within a single plant4. Hence, depending of what crop species, crop variety, or plant tissue is used for a biorefinery, the processing steps for depolymerisation by chemical/enzymatic processes and subsequent fermentation of the various sugars to liquid biofuels need to be adjusted and optimized. This fact underpins the need for a thorough characterization of plant biomass feedstocks. Here we describe a comprehensive analytical methodology that enables the determination of the composition of lignocellulosics and is amenable to a medium to high-throughput analysis (Figure 1). The method starts of with preparing destarched cell wall material. The resulting lignocellulosics are then split up to determine its monosaccharide composition of the hemicelluloses and other matrix polysaccharides1, and its content of crystalline cellulose7. The protocol for analyzing the lignin components in lignocellulosic biomass is discussed in Part I3.

Watch this Scientific Journal Video about Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part II: Carbohydrates at JoVE.com

Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part II: Carbohydrates

Plant biomass is a major carbon-neutral renewable resource that could be used for the production of biofuels. Plant biomass consists mainly of cell walls, a structurally complex composite material termed lignocellulosics. Here we describe a protocol for a comprehensive analysis of the content and composition of wall derived carbohydrates.

Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part II: Carbohydrates

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st ...

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Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 1

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 1 of 3.

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells.  Members of ...

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 2

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 2 of 3.

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 3

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 3 describes the process of fluorescently labeling the amplified RNA from both host and viral samples by amino allyl coupling of dyes. Part 3 of 3.

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristics, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004 and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. As described in Part I: Digestion and Collection of Pancreatic Tissue, human pancreas was trimmed, cannulated, perfused, and digested. After collection and at least 30 minutes of incubation in UW solution, the tissue was loaded in the cell separator (COBE 2991, Cobe, Lakewood, CO) for purification 3. Following purification, islet yield (expressed as islet equivalents, IEQ), tissue volume, and purity was determined according to standard methods 9. Isolated islets were cultured in CMRL-1066 media (Mediatech, Herndon, VA), supplemented with 1.5% human albumin, 0.1% insulin-transferrin-selenium (ITS), 1 ml of Ciprofloxacin, 5 ml o f 1M HEPES, and 14.5 ml of 7.5% Sodium Bicarbonate in T175 flasks at 37&deg;C overnight culture before islets were transplanted or used for research.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part II: purification and culture of human islets) using a modified automated method.

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part I: Lignin

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st century. This has rekindled interest in the use of plant products as industrial raw materials for the production of liquid fuels for transportation1 and other products such as biocomposite materials7. Plant biomass remains one of the greatest untapped reserves on the planet4. It is mostly comprised of cell walls that are composed of energy rich polymers including cellulose, various hemicelluloses (matrix polysaccharides, and the polyphenol lignin6 and thus sometimes termed lignocellulosics. However, plant cell walls have evolved to be recalcitrant to degradation as walls provide tensile strength to cells and the entire plants, ward off pathogens, and allow water to be transported throughout the plant; in the case of trees up to more the 100 m above ground level. Due to the various functions of walls, there is an immense structural diversity within the walls of different plant species and cell types within a single plant4. Hence, depending of what crop species, crop variety, or plant tissue is used for a biorefinery, the processing steps for depolymerization by chemical/enzymatic processes and subsequent fermentation of the various sugars to liquid biofuels need to be adjusted and optimized. This fact underpins the need for a thorough characterization of plant biomass feedstocks. Here we describe a comprehensive analytical methodology that enables the determination of the composition of lignocellulosics and is amenable to a medium to high-throughput analysis. In this first part we focus on the analysis of the polyphenol lignin (Figure 1). The method starts of with preparing destarched cell wall material. The resulting lignocellulosics are then split up to determine its lignin content by acetylbromide solubilization3, and its lignin composition in terms of its syringyl, guaiacyl- and p-hydroxyphenyl units5. The protocol for analyzing the carbohydrates in lignocellulosic biomass including cellulose content and matrix polysaccharide composition is discussed in Part II2.

Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part I: Lignin

Plant biomass is a major carbon-neutral renewable resource that could be used for the production of biofuels. Plant biomass consists mainly of cell walls, a structurally complex composite material termed lignocellulosics. Here we describe a protocol for a comprehensive analysis of the content and composition of the polyphenolic lignin.

Label-free in situ Imaging of Lignification in Plant Cell Walls

Meeting growing energy demands safely and efficiently is a pressing global challenge. Therefore, research into biofuels production that seeks to find cost-effective and sustainable solutions has become a topical and critical task. Lignocellulosic biomass is poised to become the primary source of biomass for the conversion to liquid biofuels1-6. However, the recalcitrance of these plant cell wall materials to cost-effective and efficient degradation presents a major impediment for their use in the production of biofuels and chemicals4. In particular, lignin, a complex and irregular poly-phenylpropanoid heteropolymer, becomes problematic to the postharvest deconstruction of lignocellulosic biomass. For example in biomass conversion for biofuels, it inhibits saccharification in processes aimed at producing simple sugars for fermentation7. The effective use of plant biomass for industrial purposes is in fact largely dependent on the extent to which the plant cell wall is lignified. The removal of lignin is a costly and limiting factor8 and lignin has therefore become a key plant breeding and genetic engineering target in order to improve cell wall conversion.
Analytical tools that permit the accurate rapid characterization of lignification of plant cell walls become increasingly important for evaluating a large number of breeding populations. Extractive procedures for the isolation of native components such as lignin are inevitably destructive, bringing about significant chemical and structural modifications9-11. Analytical chemical in situ methods are thus invaluable tools for the compositional and structural characterization of lignocellulosic materials. Raman microscopy is a technique that relies on inelastic or Raman scattering of monochromatic light, like that from a laser, where the shift in energy of the laser photons is related to molecular vibrations and presents an intrinsic label-free molecular "fingerprint" of the sample. Raman microscopy can afford non-destructive and comparatively inexpensive measurements with minimal sample preparation, giving insights into chemical composition and molecular structure in a close to native state. Chemical imaging by confocal Raman microscopy has been previously used for the visualization of the spatial distribution of cellulose and lignin in wood cell walls12-14. Based on these earlier results, we have recently adopted this method to compare lignification in wild type and lignin-deficient transgenic Populus trichocarpa (black cottonwood) stem wood15. Analyzing the lignin Raman bands16,17 in the spectral region between 1,600 and 1,700 cm-1, lignin signal intensity and localization were mapped in situ. Our approach visualized differences in lignin content, localization, and chemical composition. Most recently, we demonstrated Raman imaging of cell wall polymers in Arabidopsis thaliana with lateral resolution that is sub-&mu;m18. Here, this method is presented affording visualization of lignin in plant cell walls and comparison of lignification in different tissues, samples or species without staining or labeling of the tissues.

Label-free in situ Imaging of Lignification in Plant Cell Walls

A method based on confocal Raman microscopy is presented that affords label-free visualization of lignin in plant cell walls and comparison of lignification in different tissues, samples or species.

Meeting growing energy demands safely and efficiently is a pressing global challenge. Therefore, research into biofuels production that seeks to find ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...