This work was supported by grants from the St, Joseph&#8217;s Foundation (AJF, LJI) and American Lung Association, RG-224607-N (LJI).

1. Cell Line Maintenance
<ol>
	<li>Immortalized Human BE high-grade dysplastic (CP-B, CP-C, CP-D) and metaplastic (CP-A) cell lines by stable transfection of the human telomerase reverse transcriptase (TERT) protein11.</li>
	<li>Maintain BE cell lines in keratinocyte media supplemented with bovine pituitary extract (BPE), epidermal growth factor (EGF), 5% fetal bovine serum (FBS), nonessential amino acids (NEAA), penicillin-streptomycin-neomycin (PSN), and standard tissue culture conditions (37 &#176;C, 5% CO2, 98% humidity).</li>
</ol>
2. BE Cell Plating
<ol>
	<li>Preparation of 12-well plate/coverslips. Place 18 mm coverslips into a glass Petri dish and autoclave. Transfer autoclaved 18 mm glass coverslips into a 12-well tissue culture dish using a sterile glass pipette attached to a vacuum tube. Wash the coverslip with sterile PBS and media.</li>
	<li>Trypsinize and count immortalized BE cells. Count cells using an automated cell counter or a hemocytometer. Dilute cells in culture media to a concentration of 20,000-40,000 cells/ml.</li>
	<li>Place 1 ml of cells into each well containing a coverslip for a total cell count of 20,000-40,000 cells/well.</li>
	<li>Use a sterile pipette tip to gently push the coverslip to the bottom of the well to ensure that cells attach to the coverslip.</li>
</ol>
3. Drug Treatment of BE Cells
<ol>
	<li>Dilute selected drug in warm (37 &#176;C) media to desired concentration and mix well by inverting the tube repeatedly or using a vortex mixer. Determined drug concentrations empirically. Begin treatment 24 hr following the initial seeding of the BE cells to allow cells attachment on to the coverslip</li>
	<li>For comparison and control, treat an additional coverslip of BE cells with 0.1% vehicle (dimethyl sulfoxide [DMSO] or agent used to solubilize compound) diluted in culture media. Label plate with a lab pen to identify drug and vehicle treated samples. Place cells into a cell culture incubator set to 37 &#176;C, 5% CO2, 98% humidity and incubate for desired time points.</li>
</ol>
4. Immunofluorescent Staining 
<ol>
	<li>Fixation
	<ol>
		<li>Following desired incubation time of drug treatment, aspirate the media and wash the coverslips quickly with 1x phosphate buffered saline (PBS) at room temperature (RT, 25 &#176;C).</li>
		<li>Aspirate the PBS and add 1 ml of PBS containing 4% PFA [4% PFA/PBS] to each well and incubate at RT for 30 min.</li>
		<li>Aspirate the 4% PFA/PBS and wash the coverslips 3x, 5 min each with PBS at RT. Store coverslips for one week in PBS/0.1% PFA at 4 &#176;C if desired.</li>
	</ol>
	</li>
	<li>Permeabilization/Blocking
	 Permeabilize fixed PFA cells to allow the antibody to access the intracellular targets. For extracellular surfaced proteins, perform all subsequent steps without saponin added to the 1x PBS/0.5% BSA solution.
	<ol>
		<li>Reduce background by incubating with 50 mM NH4Cl diluted in PBS for 10 min at RT and wash with RT PBS once for 5 min.</li>
		<li>Incubate BE cells for 30 min in 100-300 &#956;l of PBS containing 0.1% saponin and 0.5% bovine serum albumin (BSA). Dilute saponin from filter-sterilized 10% stock, prepared in water and stored at 4 &#176;C.</li>
	</ol>
	NOTE: This prevents nonspecific binding of antibodies to cellular proteins by blocking coverslips due to the addition of 0.5% BSA. Alternatively, substitute BSA with goat serum diluted to 5% to help reduce nonspecific binding of antibodies.
	</li>
	<li>Preparation of Human Chamber
	<ol>
		<li>Prepare a square bioassay dish by layering the bottom of the chamber with wet paper towels and place a layer of Parafilm on top.</li>
		<li>Gently transfer the coverslips, cells facing upward, onto the Parafilm using forceps and a syringe needle.</li>
		<li>Mark the Parafilm with a lab pen to identify coverslips.</li>
	</ol>
	</li>
	<li>Primary Antibody Incubation
	<ol>
		<li>Dilute the primary antibody in PBS containing both 0.5% BSA and 0.1% saponin.</li>
		<li>Gently blot off the blocking solution using a laboratory tissue and add 100 &#956;l of the primary antibody solution. Incubate the primary antibody on the BE cells overnight at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Secondary Antibody Incubation
	<ol>
		<li>Wash the slides 3x with PBS/0.5% BSA/0.1% saponin for 5 min each at RT.</li>
		<li>Dilute secondary antibody conjugated with a fluorescent tag in PBS/0.5% BSA/0.1% saponin that the secondary antibody recognizes the species in which the primary antibody was raised.</li>
		<li>Add 100 &#956;l of diluted antibody to each coverslip. Incubate the secondary antibody for 2 hr at RT.</li>
	</ol>
	</li>
	<li>Washing and Mounting of Coverslips
	<ol>
		<li>Wash the coverslips 3x, 5 min each in PBS/0.5% BSA/0.1% saponin. Remove the wash buffer and add 100-300 &#956;l of PBS to the coverslips. 
		NOTE: For double antibody staining, repeat steps 4.4-4.5 with the desired additional primary and secondary antibody according to information outlined in the discussion.</li>
		<li>Using forceps, gently lift up the coverslip and blot off the PBS using a laboratory tissue or paper towel.</li>
		<li>Add 15 &#956;l of a suitable anti-fade mounting media containing 4&#39;,6-diamidino-2-phenylindole (DAPI) to the coverslip and invert the coverslip, cells down, onto a glass microscope slide.</li>
		<li>Place the slides into a slide folder and incubate away from direct light overnight at RT to allow the hard set anti-fade medium to cure, according to manufacturer&#8217;s instructions. Once the anti-fade medium has set, slides can be stored at either 4 &#176;C or -20 &#176;C until microscopic visualization.</li>
	</ol>
	</li>
</ol>
5. Microscopic Visualization of Coverslips
Stained cells can be imaged via either confocal microscopy or an IF capable upright microscope. Although confocal microscopy provides high-resolution images at discrete depths, an upright microscopic is capable of producing useful images faster. For brevity, a method of imaging stained BE cells using standard microscopy is presented. In general, to image fluorescein isothiocyanate (FITC) or similar fluorophores (i.e. green fluorescent protein), Texas Red and DAPI, requires a microscope with filters capable of discriminating these fluorophores. Check the microscope before beginning protocol to determine compatible fluorophores.
<ol>
	<li>Imaging on a Standard IF Microscope
	<ol>
		<li>Remove stored slides/coverslips from -20 &#176;C or 4 &#176;C and allow them to warm to room temperature. Power on microscope and mercury arc lamp.</li>
		<li>Place microscope slide with coverslip facing towards objective onto microscope stage. For use of an oil immersion objective, add a drop of immersion oil onto coverslip.</li>
		<li>Select the DAPI filter and open the shutter.</li>
		<li>Focus the objective while looking through the eyepieces until the image appears. Since all cells will stain with DAPI, the nuclei should be easily observable.</li>
		<li>Collect images using respective image processing software.</li>
	</ol>
	</li>
</ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+adenocarcinoma+of+the+esophagus+among+white+Americans+by+sex,+stage,+and+age.">Incidence of adenocarcinoma of the esophagus among white Americans by sex, stage, and age.</a> J. Natl. Cancer Inst. 100, 1184-1187 (2008).</li><li>Konturek, P. C., Burnat, G., Hahn, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+Barret's+adenocarcinoma+cell+growth+by+simvastatin:+involvement+of+COX-2+and+apoptosis-related+proteins.">Inhibition of Barret's adenocarcinoma cell growth by simvastatin: involvement of COX-2 and apoptosis-related proteins.</a> J. Physiol. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+small+molecule+inhibitor+of+the+Src+kinase,+reduces+the+growth+and+activates+apoptosis+in+pre-neoplastic+Barrett's+esophagus+cell+lines:+evidence+for+a+noninvasive+treatment+of+high-grade+dysplasia.">a small molecule inhibitor of the Src kinase, reduces the growth and activates apoptosis in pre-neoplastic Barrett's esophagus cell lines: evidence for a noninvasive treatment of high-grade dysplasia.</a> Thoracic Cardiovasc. Surg. 145, 531-538 (2013).</li><li>Jovov, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+transport+and+barrier+function+in+a+telomerase-immortalized+human+nondysplastic,+Barrett's+cell+line+(BAR-T).">Ion transport and barrier function in a telomerase-immortalized human nondysplastic, Barrett's cell line (BAR-T).</a> Dis. Esophagus. 22, 386-395 (2009).</li><li>Merlo, L. M., Kosoff, R. E., Gardiner, K. L., Maley, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+co-culture+model+of+esophageal+cells+identifies+ascorbic+acid+as+a+modulator+of+cell+competition.">An in vitro co-culture model of esophageal cells identifies ascorbic acid as a modulator of cell competition.</a> BMC Cancer. 11, 461 (2011).</li><li>Zhang, H. Y., Hormi-Carver, K., Zhang, X., Spechler, S. J., Souza, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+benign+Barrett's+epithelial+cells,+acid+exposure+generates+reactive+oxygen+species+that+cause+DNA+double-strand+breaks.">In benign Barrett's epithelial cells, acid exposure generates reactive oxygen species that cause DNA double-strand breaks.</a> Cancer Res. 69, 9083-9089 (2009).</li><li>Palanca-Wessels, M. C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extended+lifespan+of+Barrett's+esophagus+epithelium+transduced+with+the+human+telomerase+catalytic+subunit:+a+useful+in+vitro+model.">Extended lifespan of Barrett's esophagus epithelium transduced with the human telomerase catalytic subunit: a useful in vitro model.</a> Carcinogenesis. 24, 1183-1190 (2003).</li><li>Kumble, S., Omary, M. B., Cartwright, C. A., Triadafilopoulos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Src+activation+in+malignant+and+premalignant+epithelia+of+Barrett's+esophagus.">Src activation in malignant and premalignant epithelia of Barrett's esophagus.</a> Gastroenterology. 112, 348-356 (1997).</li><li>Inge, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=alpha-Catenin+overrides+Src-dependent+activation+of+beta-catenin+oncogenic+signaling.">alpha-Catenin overrides Src-dependent activation of beta-catenin oncogenic signaling.</a> Mol. Cancer Ther. 7, 1386-1397 (2008).</li><li>Coluccia, A. M. 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The authors declare that they have no competing financial interests.

We have outlined a method for application of IF of BE cells towards elucidating the physiological effects of targeted therapies upon these cells. While we have described the use of these procedures towards BE cells, we have found that these methods are also applicable to a variety of different cell types13,17,18. Further, these procedures can be altered in several ways to optimize staining for specific targets.
We find one parameter that has a direct effect upon proper IF is the cho...

Barrett&#8217;s esophagus (BE) is a metaplastic change in the normal squamous epithelium of the esophagus and a consequence of chronic exposure to the gastric contents resulting from gastroesophageal reflux disease (GERD)1. BE is thought to be a protective mechanism in response to GERD, however the presence of BE imparts an increased risk of esophageal adenocarcinoma (EAC), a disease which carries a significantly poor survival1. Current estimates suggest that up to 5.6% of the American population have BE, however as BE is often asymptomatic, it is thought that the majority of BE remains undiagnosed2. As incidence rates of both GERD and EAC have seen continued growth3, it has become important to understand the molecular mechanisms involved in progression of BE to EAC, particularly as this information could potentially provide therapeutic approaches towards preventing progression of BE to EAC.
Patient directed studies have resulted in the current paradigm of BE-EAC pathogenesis1. Chronic inflammation, injury, and genotoxic damage resulting from prolonged exposure to gastric contents exert selective pressure on the BE lesion promoting neoplastic progression to EAC. A number of genetic alterations have been identified during BE to EAC progression. However, despite this there is a distinct lack of understanding about the exact changes in cell signaling and subsequent effects upon cell structure and function.
Immortalized in vitro cell lines are useful tools for studying the effects of cell signaling, particularly in investigating the effects of targeted therapeutic compounds. The recent commercial availability of immortalized BE cell lines allows for such studies. Although high-throughput assays, such as the widely used viability assays, can be valuable in assessing the effects of targeted therapies upon cell proliferation and survival4-6, these assays are not useful for interrogating the effects of cell signaling upon cell morphology. Immunofluorescent staining (IF) is a useful technique for investigating the effects of targeted therapies upon the morphological, growth, and survival characteristics of cells and the proteins that are involved. Our laboratory has adapted these methods towards immortalized BE cell lines, using IF to evaluate the effects of a clinically available drug upon BE cell lines in hopes of delineating a possible chemopreventative treatment and biomarker for drug treatment7. Similarly, application of these techniques in EAC, BE and immortalized esophageal cell lines has delineated critical findings regarding BE to EAC progression8-10. We find IF analysis of BE cells treated with targeted therapies has value, allowing for characterization of changes in cell structure and protein localization. Here, we present our methods for IF of immortalized BE cells to characterize drug treatment.

<table border="0" cellpadding="0" cellspacing="0" width="743">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Name of the Material/Equipment</td>
			<td style="width:116px;">Company</td>
			<td style="width:108px;">Catalog Number</td>
			<td style="width:224px;">Comments/ Description (optional)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CP-A (Metaplastic Cell Line)</td>
			<td>ATCC</td>
			<td>CRL-4027</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CP-B (High-grade Dysplastic Cell Line)</td>
			<td>ATCC</td>
			<td>CRL-4028</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CP-C (High-grade Dysplastic Cell Line)</td>
			<td>ATCC</td>
			<td>CRL-4029</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CP-D (High-grade Dysplastic Cell Line)</td>
			<td>ATCC</td>
			<td>CRL-4030</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Keratinocyte-SFM (1X), Liquid</td>
			<td>Life Technologies</td>
			<td>17005-042</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.25% Trypsin-EDTA (1X), Phenol Red&#160;</td>
			<td>Life Technologies</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum, Qualified, HI</td>
			<td>Life Technologies</td>
			<td>10438026</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PSN ANTIBIOTIC MIXTURE</td>
			<td>Life Technologies</td>
			<td>15640</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS - Phosphate-Buffered Saline</td>
			<td>Life Technologies</td>
			<td>10010049</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pro-long Gold Antifade Reagent with DAPI&#160;</td>
			<td>Life Technologies</td>
			<td>P36931</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Circular Glass Coverslip 18mm</td>
			<td>Fisher Scientific</td>
			<td>15-183-86</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#946;-catenin Primary Antibody (Rabbit)</td>
			<td>Cell Signaling</td>
			<td>9562S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alexafluor 488 (Goat Anti-Rabbit)</td>
			<td>Life Technologies</td>
			<td>A11008</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10cm TC treated PS dish, sterile</td>
			<td>USA Scientific</td>
			<td>CC7682-3394</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">12-well TC treated PS plate, sterile</td>
			<td>USA Scientific</td>
			<td>5666-5180</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>276855</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>A9434</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Saponin</td>
			<td>Sigma-Aldrich</td>
			<td>47036</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine Serum Albumin - Fraction V</td>
			<td>Sigma-Aldrich</td>
			<td>85040C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SKI-606 (Bosutinib)</td>
			<td>Selleck Chemicals</td>
			<td>S1014</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Square Bioassay Dish&#160;</td>
			<td>Thermo Scientific</td>
			<td>240835</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Parafilm&#160;</td>
			<td>VWR</td>
			<td>82024-546</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Disposable Pasteur Pipets, Flint Glass</td>
			<td>VWR</td>
			<td>14672-380</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nexcelom Mini Cell Counter</td>
			<td>Nexcelom</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cellometer Counting Chambers</td>
			<td>Nexcelom</td>
			<td>CHT4-SD100-014</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Zeiss Apotome microscope&#160;</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

an immunofluorescent method for characterization of barrett&#8217;s esophagus cells

Esophageal adenocarcinoma (EAC) has an overall survival rate of less than 17% and incidence of EAC has risen dramatically over the past two decades. One of the primary risk factors of EAC is Barrett&#8217;s esophagus (BE), a metaplastic change of the normal squamous esophagus in response to chronic heartburn. Despite the well-established connection between EAC and BE, interrogation of the molecular events, particularly altered signaling pathways involving progression of BE to EAC, are poorly understood. Much of this is due to the lack of suitable in vitro models available to study these diseases. Recently, immortalized BE cell lines have become commercially available allowing for in vitro studies of BE. Here, we present a method for immunofluorescent staining of immortalized BE cell lines, allowing in vitro characterization of cell signaling and structure after exposure to therapeutic compounds. Application of these techniques will help develop insight into the mechanisms involved in BE to EAC progression and provide potential avenues for treatment and prevention of EAC.

An example of the results obtained from application of the described procedures is illustrated in Figures 1A-D. Coverslips with CP-D and CP-C BE cells were treated for 24 hr with 1 &#956;M of the Src family inhibitor, SKI-606, or vehicle (DMSO) and stained using the above procedures for the Adherens Junction and Wnt signaling protein, &#946;-catenin. Visualization of &#946;-catenin was achieved by labeling with an anti-rabbit IgG coupled to a green fluorophore, while labeling of the cell nucleus was acco...

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An Immunofluorescent Method for Characterization of Barrett&amp;#8217;s Esophagus Cells

There is a discernible need for improved information on molecular drivers of Barrett&#8217;s Esophagus. Immunofluorescent staining is a useful technique for understanding the effects of cell signaling on cell morphology. We present a simple, effective protocol for the use of immunofluorescent staining to assess therapeutic treatment in Barrett&#8217;s Esophagus cells.

An Immunofluorescent Method for Characterization of Barrett&#8217;s Esophagus Cells

Esophageal adenocarcinoma (EAC) has an overall survival rate of less than 17% and incidence of EAC has risen dramatically over the past two decades. One ...

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JoVE Journal

Biology

14.0K ビュー.  St. Joseph's Hospital and Medical Center. バレット食道の分子ドライバーを改良し、情報のための識別可能な必要性がある。免疫蛍光染色は、細胞形態学上の細胞シグナル伝達の効果を理解するために有用な技術である。私たちは、バレット食道細胞において治療上の処置を評価するための免疫蛍光染色を使用するためのシンプルで効果的なプロトコルを提示する。 

ビデオ: An Immunofluorescent Method for Characterization of Barrett&#8217;s Esophagus Cells

A Method For Production of Recombinant mCD1d Protein in Insect Cells.

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated heavy chains that are noncovalently associated with beta2-microglobulin. They bind lipids rather than peptides. Although their structure confirms the similarity of CD1 proteins to MHC class I and class II antigen presenting molecules, the mCD1d groove is relatively narrow, deep, and highly hydrophobic and it has two binding pockets instead of the several shallow pockets described for the classical MHC-encoded antigen-presenting molecules. Based upon their amino acid sequences, such a hydrobphobic groove provides an ideal environment for the binding of lipid antigens. The Natural Killer T (NKT) cells use their TCR to recognize glycolipids bound to or presented by CD1d. T cells reactive to lipids presented by CD1 have been involved in the protection against autoimmune and infectious diseases and in tumor rejection. Thus, the ability to identify, purify , and track the response of CD1-reactive NKT cell is of great importance . The generation of tetramers of alpha Galactosyl ceramide (a-Galcer) with CD1d has significant insight into the biology of NKT cells. Tetramers constructed from other CD1 molecules have also been generated and these new reagents have greatly expanded the knowledge of the functions of lipid-reactive T cells, with potential use in monitoring the response to lipid-based vaccines and in the diagnosis of autoimmune diseases and other treatments.

A Method to prepare Insect cells and infect them with baculovirus for the the purpose of production of recombinant mCD1d proteinand generating mCD1d tetramers.

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated ...

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

An Innovative Method for Exosome Quantification and Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology.
Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. 
Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).

A method for isolation of exosomes from whole blood and further analysis by nanoparticle tracking using a semi-automatic instrument is presented in this article. The presented technology provides an extremely sensitive method for visualizing and analyzing particles in liquid suspension.

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about ...

Isolation of Myofibroblasts from Mouse and Human Esophagus

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, and subjected to enzymatic digestion with collagenase and dispase for 25 min. Human esophageal resection specimens are stripped of muscularis propria and adventitia and the remaining mucosa is minced, and subjected to enzymatic digestion with collagenase and dispase for up to 6 hr. Cultured cells express &#945;-SMA and vimentin and express desmin weakly or not at all. Culture conditions are not conducive to growth of epithelial, hematopoietic, or endothelial cells. Culture purity is further confirmed by flow cytometric evaluation of cell surface marker expression of potential contaminating hematopoietic and endothelial cells. The described technique is straightforward and results in consistent generation of non-hematopoieitc, non-endothelial stromal cells. Limitations of this technique are inherent to the use of primary cultures in molecular biology studies, i.e., the unavoidable variability encountered among cultures established across different mice or humans. Primary cultures however are a more representative reflection of the in vivo state compared to cell lines. These methods also provide investigators the ability to isolate and culture stromal cells from different clinical and experimental conditions, allowing comparisons between groups. Characterized esophageal stromal cells can also be used in functional studies investigating epithelial-stromal interactions in esophageal disorders.

We present a protocol to generate primary cultures of murine and human esophageal stromal cells with a myofibroblast phenotype. Cultured cells have spindle shaped morphology, express &#945;-SMA and vimentin, and lack epithelial, hematopoietic and endothelial cell surface markers. Characterized stromal cells can be used in functional studies of epithelial-stromal interactions.

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, ...

Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current literature describes several different methodological approaches to obtain images of intestinal tissues for data validation. In this paper, we delineate a common protocol relating to the derivation and processing of mouse intestinal tissues. Proper fixation of intestinal tissues and Swiss-roll techniques that enhance intestinal epithelial morphology are discussed. Postresection processing and reorientation of embedded intestinal tissues are critical in obtaining paraffin-embedded blocks that display intact intestinal structural features after sectioning. The Swiss-rolling technique helps in histological assessment of the complete intestinal or colonic sections examined. An ability to differentiate intestinal structural features can be vital in quantitative measurements of intestinal inflammation and tumorigenesis along the entire length. Finally, paraffin-embedded sections are ideal for robust processing using both immunohistochemical and immunofluorescent detection methods. Nonfluorescent immunohistochemical sections provide a vibrant image of the tissue detailing different cellular structural features but do not provide flexibility for intracellular co-localization experiments. Multiple fluorescent channels can be appropriately utilized with immunofluorescent detection for co-localization experiments, lending support to mechanistic studies.

Accurate identification and location of epithelial cells along the intestinal mucosal lining are essential to define different cell lineages. Proper imaging of intestinal tissues is crucial for identification of protein expression patterns with maximum resolution. This study aims to delineate the optimal methods and conditions for processing mouse intestinal tissues.

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current ...

An Easy Method for Plant Polysome Profiling

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the analysis of translational regulation. The method described here is an easy and economical way for fractionating polysomes from various plant tissues. A sucrose gradient is made without the need for a gradient maker by sequentially freezing each layer. Cytosolic extracts are then prepared in a buffer containing cycloheximide and chloramphenicol to immobilize the cytosolic and chloroplastic ribosomes to mRNA and are loaded onto the sucrose gradient. After centrifugation, six fractions are directly collected from the bottom to the top of the gradient, without piercing the ultracentrifugation tube. During collection, the absorbance at 260 nm is read continuously to generate a polysome profile that gives a snapshot of global translational activity. Fractions are then pooled to prepare three different mRNA populations: the polysomes, mRNAs bound to several ribosomes; the monosomes, mRNAs bound to one ribosome; and mRNAs that are not bound to ribosomes. mRNAs are then extracted. This protocol has been validated for different plants and tissues including Arabidopsis thaliana seedlings and adult plants, Nicotiana benthamiana, Solanum lycopersicum, and Oryza sativa leaves.

This protocol describes an easy method to extract and fractionate transcripts from plant tissues on the basis of the number of bound ribosomes. It allows a global estimate of translation activity and the determination of the translational status of specific mRNAs.

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and age-related muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Active autophagy is associated with productive muscle regeneration, which is essential for Muscle Stem Cell (MuSC) activation. Here, we provide a protocol for the in situ detection of LC3, an autophagy marker in MyoD-positive MuSCs of muscle tissue sections from control and injured mice.

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal ...

An Antegrade Perfusion Method for Cardiomyocyte Isolation from Mice

In basic research using mouse heart, isolating viable individual cardiomyocytes is a crucial technical step to overcome. Traditionally, isolating cardiomyocytes from rabbits, guinea pigs or rats has been performed via retrograde perfusion of the heart with enzymes using a Langendorff apparatus. However, a high degree of skill is required when this method is used with a small mouse heart. An antegrade perfusion method that does not use a Langendorff apparatus was recently reported for the isolation of mouse cardiomyocytes. We herein report a complete protocol for the improved antegrade perfusion of the excised heart to isolate individual heart cells from adult mice (8 - 108 weeks old). Antegrade perfusion is performed by injecting perfusate near the apex of the left ventricle of the excised heart, the aorta of which was clamped, using an infusion pump. All procedures are carried out on a pre-warmed heater mat under a microscope, which allows for the injection and perfusion processes to be monitored. The results suggest that ventricular and atrial myocytes, and fibroblasts can be well isolated from a single adult mouse simultaneously.

We developed a simple method for isolating high quality individual mouse heart cells by the antegrade perfusion technique. This method is Langendorff-free and useful for isolating ventricular and atrial myocytes or interstitial cells, such as cardiac fibroblasts or progenitors.