The project described was supported by Grant Numbers P01 AI055637 and U54 AI057156 from NIAID/NIH. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the RCE Programs Office, NIAID, or NIH.

Day 1: F. tularensis plate inoculation <ol> <li>Plate frozen stock cultures of F. tularensis onto Mueller-Hinton agar supplemented with 2.5% (vol/vol) donor calf serum, 2% (vol/vol) IsoVitaleX, 0.1% (wt/vol) glucose, and 0.025% (wt/vol) iron pyrophosphate. Grow F. tularensis at 37&deg;C with 5% CO2 for approximately 24 h.</li> </ol> Day 2: F. tularensis liquid media inoculation <ol> <li>Prepare and autoclave 1 liter of cation-adjusted Mueller-Hinton medium (containing 1.23 mM calcium chloride dihydrate and 1.03 mM magnesium chloride hexahydrate). When cooled to 37&deg;C, further supplement medium with 0.1% (wt/vol) glucose, 0.025% (wt/vol) iron pyrophosphate, and 2% (vol/vol) IsoVitaleX. Filter sterilize (0.22 &mu;) medium into two 500-ml aliquots.</li> <li>Remove 12 ml of supplemented Mueller-Hinton medium and transfer into a sterile 50-ml Falcon tube.</li> <li>Using a sterile 10-&mu;l inoculation loop, scrape a large loopful of F. tularensis growth from agar plates (from Day 1) and transfer bacteria into 12 ml of Mueller-Hinton medium (see Step 2). Pipette solution multiple times to break-up clumps and prepare homogenous bacterial suspension. Do not vortex.</li> <li>Using a sterile pipette, inoculate each 500-ml vessel with 5 ml of bacterial suspension from Step 3. Grow broth cultures at 37&deg;C for 14 to 18 h with gentle shaking (190-200 rpm on a New Brunswick Innova 2300 series shaker).</li> </ol> Day 3: Spheroplasting, osmotic lysis, and sucrose density gradient centrifugation <ol> <li>Obtain F. tularensis cultures from the shaking incubator and remove a 1-ml aliquot from each 500-ml culture to check the respective optical densities. We have found optimum membrane extraction and isolation results when using F. tularensis cells in early logarithmic phase of growth, which correlates with an OD600 between 0.2 to 0.4 (~107 to 108 CFU/ml). Cultures with an OD600 less than 0.2 will not yield sufficient quantity of total membrane material for subsequent sucrose gradients. Conversely, cultures with an OD600 greater than 0.4 (nearing stationary phase) have been found to yield mixed-membrane fusions.</li> <li>Centrifuge cultures at 7,500 x g for 30 min at 15&deg;C to collect the cells. We recommend centrifugation of cultures in four 250-ml centrifuge bottles, because it facilitates subsequent pellet suspension.</li> <li>Carefully remove the media supernatant from each centrifuge bottle and firmly tap the bottles on absorbent material to remove excess growth medium.</li> <li>Within 10 min following completion of centrifugation, suspend each bacterial pellet in 8.75 ml of 0.75 M sucrose (in 5 mM Tris, pH 7.5). All four bacterial pellets should be suspended and transferred (35 ml total of bacterial suspension) to a sterile 250-ml flask (with a small stirbar) within 10 min.</li> <li>While gently mixing the cell suspension on a stirplate, slowly add 70 ml (2 volumes) of 10 mM disodium EDTA (in 5 mM Tris, pH 7.8) over the course of 10 min. Add the EDTA solution with the tip of pipette below the cell suspension level to avoid elevated local concentrations of EDTA. The stirbar should be rotating at a rate sufficient to thoroughly mix the cell suspension but not fast enough to cause frothing or bubble formation. After the EDTA solution has been added, incubate the solution for 30 min at room temperature.</li> <li>After the 30 min incubation, slowly add 11 ml (1/10th volume) of a 2 mg/ml lysozyme solution to a final concentration of 200 &mu;g/ml. Continue to mix the cell suspension during the lysozyme solution addition. As noted above, add the lysozyme solution with the pipette tip below the cell suspension fluid level to avoid elevated local concentrations of lysozyme. Stock lysozyme solutions (2 mg/ml) can be prepared in single-use aliquots and stored at -20&deg;C for three to four months. Incubate the resulting solution for 30 min at room temperature.</li> <li>During the 30 min incubation, prepare a sterile 1L flask with a small stirbar and 530 ml of room temperature Cellgro Molecular Grade dH2O (4.5 volumes).</li> <li>Osmotically lyse the cell suspension (from Step 6) by slow dilution (flow rate of approximately 11 ml/min) into 530 ml of Molecular Grade dH2O (from Step 7) over the course of 10 to 15 min with gentle mixing. Because of the larger solution volume in this step, increase the stirbar speed to ensure proper mixing. The stirbar should be rotating at a rate sufficient to thoroughly disperse the cell suspension once added to the dH2O but not fast enough to lead to frothing or bubble formation. Add the cell suspension with the tip of pipette resting on the bottom of the flask, adjacent to the stirbar, to facilitate uniform dilution of the cell suspension. We have found that 25-ml pipettes work best during this step. Carefully monitor the dilution process and interrupt the flow rate as necessary to disperse local accumulations of cells, cell clumps, and cell wisps. Incubate the resulting solution for 30 min at room temperature. Note that this step appears to be one of the most challenging and critical for overall success of the experiment.</li> <li>After the 30 min incubation, transfer 40 ml aliquots of the osmotic lysis solution into 50-ml conical tubes and centrifuge at 7500 x g for 30 min at 10&deg;C to remove intact cells and debris.</li> <li>Following centrifugation, carefully remove 27 to 30 ml of supernatant from each conical tube and pool supernatants in a sterile 1L flask</li><li>Transfer 25 ml of pooled supernatant, each, into 16 ultracentrifuge tubes and centrifuge at 200,000 x g (44,400 rpm in F50L-8x39 FiberLite ultracentrifuge rotor) for 2 h at 4&deg;C. Note that each rotor holds 8 tubes, so this step requires two rotors and two ultracentrifuges. Alternatively, the second set of 8 tubes should be held at 4&deg;C until centrifuged.</li> <li>Within 10 min of the completion of the ultracentrifuge run, prepare modified membrane resuspension buffer. Transfer 8 ml of membrane suspension buffer (25% [wt/wt] sucrose, 5 mM Tris, 30 mM MgCl2) to a sterile tube and add 1 tablet of EDTA-free protease inhibitor cocktail and 375 U of Benzonase. Gently mix solution to dissolve protease inhibitor tablet.</li> <li>Following ultracentrifugation, carefully pour off supernatants, invert ultracentrifuge tubes on absorbent material, and firmly tap tubes to remove excess supernatant. Mark the location of each membrane pellet with a marker to aid in visualization. Gently resuspend eight membrane pellets with 600 &mu;l each of modified membrane resuspension buffer. Transfer each membrane resuspension to the next set of eight tubes, gently resuspend these eight pellets, and pool the resulting membrane resuspensions in a sterile tube. The pelleted membranes are difficult to resuspend, so continue passing the modified membrane resuspension buffer over the pellet until it peels away from the wall of the tube. It is often necessary to use the micropipette tip to scrape the membrane pellet off the tube wall and aid in the resuspension process. When pipetting, avoid frothing or bubble formation. Note that this step appears to be one of the most challenging and critical for overall success of the experiment.</li> <li>When the membrane pellets have been resuspended and removed from all 16 tubes, wash every four tubes with 600 &mu;l of modified membrane resuspension buffer. The wash should be focused around the marked area (membrane pellet) of each tube. Transfer the wash into the membrane resuspension tube. Repeat the wash step for the remaining three sets of four tubes.</li> <li>Incubate the membrane resuspension with gentle rocking for 30 min at room temperature to degrade DNA and aid in reduction of flocculent material.</li> <li>Remove a small aliquot of the membrane suspension and perform protein quantification to determine total membrane yield. We typically prepare undiluted, 1:1 diluted (in 2.5% SDS), and 1:3 diluted (in 2.5% SDS) membrane samples, heat the samples at 90&deg;C for 10 min, and use the BioRad DC Protein Assay to quantify the membrane resuspension protein concentration. Total protein yield is generally between 1.0 mg/ml and 1.6 mg/ml. The use of heat and SDS are required to release OMPs from the extracted membranes so that a more accurate protein quantification value can be obtained.</li> <li>Prepare linear sucrose gradients by layering 1.8 ml each of sucrose solutions (wt/wt, prepared in 5 mM EDTA, pH 7.5) into 14 x 95 mm Ultra-Clear ultracentrifuge tubes in the following order: 55%, 50%, 45%, 40%, 35%, 30%. Layering of sucrose gradients is easiest to perform when using a bulb pipettor, not an automated pipettor.</li> <li>Based on the membrane resuspension protein quantification (Step 16), layer 1.5 mg of membrane resuspension on top of each gradient. Each 14 x 95 mm ultracentrifuge tube has a maximum capacity of 12.5 ml and, given that 10.8 ml is accounted for by the sucrose solutions from Step 17, you should not load more than 1.7 ml of membrane resuspension per gradient. Individually weigh and carefully adjust the final weight of each sucrose gradient tube (by adding or removing membrane resuspension) so that all tubes weigh the same (&plusmn; 0.01 g).</li> <li>Transfer sucrose gradients into a SW-40Ti swinging bucket rotor and centrifuge at 256,000 x g (38,000 rpm) for a minimum of 17 h at 4&deg;C.</li> </ol> Day 4: Collection of sucrose gradient fractions <ol> <li>After 17 h of centrifugation, stop the centrifuge run, and carefully remove sucrose gradients from the SW-40Ti rotor.</li> <li>Carefully clean the bottom of the sucrose gradient tube with 70% ethanol, puncture the bottom of the tube with a 21g needle, and collect sequential 500 &mu;l fractions from the gradient into sterile microfuge tubes. Repeat for subsequent gradients. The gradient should be allowed to drip by gravity flow, so as not to disturb the sucrose gradient. However, if the dripping ceases from the bottom of the gradient tube, a small amount of pressure may be applied to the top of the tube using one's finger.</li> <li>Determine the refractive index of each sucrose gradient fraction using a refractometer and correlate the refractive index with a specific density in g/ml 1. Assess protein localization by preparing representative sucrose gradient fractions (density increments of 0.01; from 1.11 g/ml to 1.26 g/ml) for SDS-PAGE separation, transfer to nitrocellulose, and immunoblotting.</li> </ol> Representative Results When performed correctly, this procedure results in the complete separation of IM and OM vesicles from both Type A (e.g. SchuS4) and Type B (e.g. LVS) strains of F. tularensis. As shown in representative immunoblots in Figure 1, F. tularensis OMPs localize between densities of 1.17 and 1.20 g/ml. By comparison, IMPs localize between densities of 1.13 and 1.14 g/ml. <img src="/files/ftp_upload/2044/2044fig1.jpg" alt="Figure 1" /> Figure 1. Immunoblotting of F. tularensis sucrose density gradients. Sequential fractions were collected from gradients and densities (g/ml) were calculated based upon refractive indices. Proteins were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted to detect OMP and IMP fractionation. mw, prestained molecular weight standards with sizes (in kDa) noted on the left side of each blot. LVS, whole cell lysates of F. tularensis LVS. The corresponding sucrose gradient fraction densities are noted above their respective lanes. OMPs and IMPs, as noted in the left margin, were detected with polyclonal, monospecific antisera. Similar results were obtained for F. tularensis SchuS4.

<ol>
	<li>Price, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrifugation+in+density+gradients.">Centrifugation in density gradients.</a> , 335-335 (1982).</li><li>Osborn, M. J., Gander, J. E., Parisi, E., Carson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+assembly+of+the+outer+membrane+of+Salmonella+typhimurium.+Isolation+and+characterization+of+cytoplasmic+and+outer+membrane.">Mechanism of assembly of the outer membrane of Salmonella typhimurium. Isolation and characterization of cytoplasmic and outer membrane.</a> J Biol Chem. 247 (12), 3962-3962 (1972).</li><li>Nikaido, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+outer+membranes.">Isolation of outer membranes.</a> Methods Enzymol. 235, 225-225 (1994).</li><li>Mizuno, T., Kageyama, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+and+characterization+of+the+outer+membrane+of+Pseudomonas+aeruginosa.">Separation and characterization of the outer membrane of Pseudomonas aeruginosa.</a> J Biochem. 84 (1), 179-179 (1978).</li><li>Huntley, J. F., Conley, P. G., Hagman, K. E., Norgard, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Francisella+tularensis+outer+membrane+proteins.">Characterization of Francisella tularensis outer membrane proteins.</a> J Bacteriol. 189, 561-561 (2007).</li><li>Huntley, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Native+outer+membrane+proteins+protect+mice+against+pulmonary+challenge+with+virulent+type+A+Francisella+tularensis.">Native outer membrane proteins protect mice against pulmonary challenge with virulent type A Francisella tularensis.</a> Infect Immun. 76 (8), 3664-3664 (2008).</li><li>Bevanger, L., Maeland, J. A., Naess, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agglutinins+and+antibodies+to+Francisella+tularensis+outer+membrane+antigens+in+the+early+diagnosis+of+disease+during+an+outbreak+of+tularemia.">Agglutinins and antibodies to Francisella tularensis outer membrane antigens in the early diagnosis of disease during an outbreak of tularemia.</a> J Clin Microbiol. 26 (3), 433-433 (1988).</li><li>Hubalek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+proteome+database+of+Francisella+tularensis.">Towards proteome database of Francisella tularensis.</a> J Chromatogr B Analyt Technol Biomed Life Sci. 787 (1), 149-149 (2003).</li><li>Pavkova, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Francisella+tularensis+live+vaccine+strain:+proteomic+analysis+of+membrane+proteins+enriched+fraction.">Francisella tularensis live vaccine strain: proteomic analysis of membrane proteins enriched fraction.</a> Proteomics. 5 (9), 2460-2460 (2005).</li><li>Sandstrom, G., Tarnvik, A., Wolf-Watz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunospecific+T-lymphocyte+stimulation+by+membrane+proteins+from+Francisella+tularensis.">Immunospecific T-lymphocyte stimulation by membrane proteins from Francisella tularensis.</a> J Clin Microbiol. 25 (4), 641-641 (1987).</li><li>Sjostedt, A., Tarnvik, A., Sandstrom, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+T-cell-stimulating+17-kilodalton+protein+of+Francisella+tularensis+LVS+is+a+lipoprotein.">The T-cell-stimulating 17-kilodalton protein of Francisella tularensis LVS is a lipoprotein.</a> Infect Immun. 59 (9), 3163-3163 (1991).</li><li>Twine, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Francisella+tularensis+proteome:+low+levels+of+ASB-14+facilitate+the+visualization+of+membrane+proteins+in+total+protein+extracts.">Francisella tularensis proteome: low levels of ASB-14 facilitate the visualization of membrane proteins in total protein extracts.</a> J Proteome Res. 4 (5), 1848-1848 (2005).</li></ol>

No conflicts of interest declared.

This protocol describes a variation of the spheroplasting, osmotic lysis, and sucrose density gradient centrifugation "gold standard" for the physical separation and enrichment of Gram-negative bacterial OMs from other cellular components 2, 3, 4. Whereas we previously described the application of this method for isolating OMs from both Type A and Type B strains of F. tularensis 5, this presentation offers a more detailed and optimized procedure for OM isolation. Indeed, this is an important advance for identifying potentially surface-exposed and presumptive virulence factors from this deadly pathogen. The significance of this procedure was demonstrated in studies by our lab showing that immunization of mice with purified OMPs afforded substantial protection against virulent Type A F. tularensis pulmonary challenge 6. As noted above, growth phase of the bacterial cells, careful monitoring during osmotic lysis, and gentle resuspension of membrane pellets are critical steps that appear to correlate with success/failure of this membrane separation procedure. While this optimized method is rigorous and subject to some experimental variability, the resulting OMs appear to be of remarkably enhanced purity as compared to those garnered from the use of detergents, lithium chloride, or sodium carbonate 7, 8, 9, 10, 11, 12.

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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>Donor calf serum</td>
<td>&nbsp;</td>
<td>Mediatech</td>
<td>35-022-CV</td>
<td>&nbsp;</td>
<tr>
<td>IsoVitaleX</td>
<td>&nbsp;</td>
<td>Becton Dickinson</td>
<td>211876</td>
<td>&nbsp;</td>
<tr>
<td>Cellgro molecular grade dH2O</td>
<td>&nbsp;</td>
<td>Mediatech</td>
<td>46-000-CM</td>
<td>&nbsp;</td>
<tr>
<td>38.5 ml polycarbonate bottle</td>
<td>&nbsp;</td>
<td>FiberLite</td>
<td>010-0514</td>
<td>&nbsp;</td>
<tr>
<td>F50L-8x39 ultracentrifuge rotor</td>
<td>&nbsp;</td>
<td>FiberLite</td>
<td>096-087051</td>
<td>&nbsp;</td>
<tr>
<td>Complete mini EDTA-free protease inhibitor cocktail</td>
<td>&nbsp;</td>
<td>Roche Diagnostics</td>
<td>04693132001</td>
<td>&nbsp;</td>
<tr>
<td>Benzonase</td>
<td>&nbsp;</td>
<td>Novagen</td>
<td>70664-3</td>
<td>&nbsp;</td>
<tr>
<td>DC protein assay</td>
<td>&nbsp;</td>
<td>BioRad</td>
<td>500-0116</td>
<td>&nbsp;</td>
<tr>
<td>Ultra-Clear 14x95mm centrifuge tubes</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>344060</td>
<td>&nbsp;</td>
<tr>
<td>SW-40Ti swinging bucket rotor</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>331301</td>
<td>&nbsp;</td>
<tr>
<td>Abbe benchtop refractometer</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>13-964-10 </td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

method for the isolation of francisella tularensis outer membranes

Francisella tularensis is a Gram-negative intracellular coccobacillus and the causative agent of the zoonotic disease tularemia. When compared with other bacterial pathogens, the extremely low infectious dose (&lt;10 CFU), rapid disease progression, and high morbidity and mortality rates suggest that the virulent strains of Francisella encode for novel virulence factors. Surface-exposed molecules, namely outer membrane proteins (OMPs), have been shown to promote bacterial host cell binding, entry, intracellular survival, virulence and immune evasion. The relevance for studying OMPs is further underscored by the fact that they can serve as protective vaccines against a number of bacterial diseases. Whereas OMPs can be extracted from gram-negative bacteria through bulk membrane extraction techniques, including sonication of cells followed by centrifugation and/or detergent extraction, these preparations are often contaminated with periplasmic and/or cytoplasmic (inner) membrane (IM) contaminants. For years, the "gold standard" method for the biochemical and biophysical separation of gram-negative IM and outer membranes (OM) has been to subject bacteria to spheroplasting and osmotic lysis, followed by sucrose density gradient centrifugation. Once layered on a sucrose gradient, OMs can be separated from IMs based on the differences in buoyant densities, believed to be predicated largely on the presence of lipopolysaccharide (LPS) in the OM. Here, we describe a rigorous and optimized method to extract, enrich, and isolate F. tularensis outer membranes and their associated OMPs.

Watch this Scientific Journal Video about Method for the Isolation of Francisella tularensis Outer Membranes at JoVE.com

Method for the Isolation of Francisella tularensis Outer Membranes

A protocol for separating inner and outer membranes from Francisella tularensis by spheroplasting, osmotic lysis, and sucrose density gradient ultracentrifugation.

Method for the Isolation of Francisella tularensis Outer Membranes

Francisella tularensis is a Gram-negative intracellular coccobacillus and the causative agent of the zoonotic disease tularemia. When compared with other ...

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17.0K Aufrufe.  University of Texas Southwestern Medical Center. Ein Protokoll für die Trennung von innerer und äußerer Membranen aus Francisella tularensis Durch Spheroplasting, osmotische Lyse und Saccharose-Dichtegradienten-Ultrazentrifugation.

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Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.

A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and ...

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

Isolation of Neonatal Extrahepatic Cholangiocytes

The intra and extrahepatic bile ducts of the liver are developmentally distinct, and may be differentially affected by certain diseases. However, differences between intra and extrahepatic cholangiocytes, and between neonatal and adult cells, are not well understood.
Methods for the isolation of cholangiocytes from intrahepatic bile ducts are well established1-4. Isolation of extrahepatic ductal cells, especially from the neonate, has not yet been described, although this would be of great benefit in understanding the differences between distinct cholangiocyte populations and in studying diseases such as biliary atresia that appear to target the extrahepatic ducts. Described here is an optimized technique to isolate both neonatal and adult mouse extrahepatic bile duct cells. This technique yields a pure cell population with minimal contamination from mesenchymal cells like fibroblasts.
This method is based on the removal of the extrahepatic ducts and gallbladder, followed by meticulous dissection and scraping to remove fat and fibroblast layers. Structures are embedded in thick layers of collagen and cultured for approximately 3 weeks to allow outgrowth of cholangiocytes in monolayers, which can then be trypsinized and re plated for experimental use.

A technique to isolate cholangiocytes from the extrahepatic bile ducts of neonatal mice is described. The ducts are meticulously dissected, and then cells are isolated by outgrowth in thick collagen gels. This method provides a useful tool for studying extrahepatic bile duct development and pathology.

The intra and extrahepatic bile ducts of the liver are developmentally distinct, and may be differentially affected by certain diseases. However, ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation.
Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

The extracellular matrix plays a major role in defining the microenvironment of cells and in modulating cell behavior and phenotype. We describe a rapid method for the isolation of cell-derived extracellular matrix, which can be adapted to different scales for microscopic, biochemical, proteomic, or functional studies.

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and ...

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of microbial contaminants present on the seed surface. Currently, Arabidopsis seeds are sterilized using two distinct sterilization techniques in conditions that differ slightly between labs and have not been standardized, often resulting in only partially effective sterilization or in excessive seed mortality. Most of these methods are also not easily scalable to a large number of seed lines of diverse genotypes. As technologies for high-throughput analysis of Arabidopsis continue to proliferate, standardized techniques for sterilizing large numbers of seeds of different genotypes are becoming essential for conducting these types of experiments. The response of a number of Arabidopsis lines to two different sterilization techniques was evaluated based on seed germination rate and the level of seed contamination with microbes and other pathogens. The treatments included different concentrations of sterilizing agents and times of exposure, combined to determine optimal conditions for Arabidopsis seed sterilization. Optimized protocols have been developed for two different sterilization methods: bleach (liquid-phase) and chlorine (Cl2) gas (vapor-phase), both resulting in high seed germination rates and minimal microbial contamination. The utility of these protocols was illustrated through the testing of both wild type and mutant seeds with a range of germination potentials. Our results show that seeds can be effectively sterilized using either method without excessive seed mortality, although detrimental effects of sterilization were observed for seeds with lower than optimal germination potential. In addition, an equation was developed to enable researchers to apply the standardized chlorine gas sterilization conditions to airtight containers of different sizes. The protocols described here allow easy, efficient, and inexpensive seed sterilization for a large number of Arabidopsis lines.

The objective of this study was to determine the effects of bleach and chlorine gas sterilization on seed germination of a range of Arabidopsis genotypes grown on sterile media. Optimized sterilization protocols have been developed to prevent the growth of microbial contaminants while providing satisfactory seed survival.

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes

Here we report a protocol to investigate the heat transfer between irradiated gold nanoparticles (GNPs) and bilayer lipid membranes by electrochemistry using tethered bilayer lipid membranes (tBLMs) assembled on gold electrodes. Irradiated modified GNPs, such as streptavidin-conjugated GNPs, are embedded in tBLMs containing target molecules, such as biotin. By using this approach, the heat transfer processes between irradiated GNPs and model bilayer lipid membrane with entities of interest are mediated by a horizontally focused laser beam. The thermal predictive computational model is used to confirm the electrochemically induced conductance changes in the tBLMs. Under the specific conditions used, detecting heat pulses required specific attachment of the gold nanoparticles to the membrane surface, while unbound gold nanoparticles failed to elicit a measurable response. This technique serves as a powerful detection biosensor which can be directly utilized for the design and development of strategies for thermal therapies that permits optimization of the laser parameters, particle size, particle coatings and composition.

This work outlines a protocol to achieve dynamic, non-invasive monitoring of heat transfer from laser-irradiated gold nanoparticles to tBLMs. The system combines impedance spectroscopy for the real-time measurement of conductance changes across the tBLMs, with a horizontally focused laser beam that drives gold nanoparticle illumination, for heat production.

Here we report a protocol to investigate the heat transfer between irradiated gold nanoparticles (GNPs) and bilayer lipid membranes by electrochemistry ...