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>

<ol><li>Reid, B. J., Li, X., Galipeau, P. C., Vaughan, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Barrett%27s+oesophagus+and+oesophageal+adenocarcinoma%3A+time+for+a+new+synthesis%5BTitle%5D)+AND+%22Nat.+Rev.+Cancer%22%5BJournal%5D)">Barrett's oesophagus and oesophageal adenocarcinoma: time for a new synthesis.</a> Nat. Rev. Cancer. 10, 87-101 (2010).</li>
<li>Hayeck, T. J., Kong, C. Y., Spechler, S. J., Gazelle, G. S., Hur, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+prevalence+of+Barrett%27s+esophagus+in+the+US%3A+estimates+from+a+simulation+model+confirmed+by+SEER+data%5BTitle%5D)+AND+%22Dis.+Esophagus%22%5BJournal%5D)">The prevalence of Barrett's esophagus in the US: estimates from a simulation model confirmed by SEER data.</a> Dis. Esophagus. 23, 451-457 (2010).</li>
<li>Brown, L. M., Devesa, S. S., Chow, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Incidence+of+adenocarcinoma+of+the+esophagus+among+white+Americans+by+sex%2C+stage%2C+and+age%5BTitle%5D)+AND+%22J.+Natl.+Cancer+Inst%22%5BJournal%5D)">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/pubmed?term=((Inhibition+of+Barret%27s+adenocarcinoma+cell+growth+by+simvastatin%3A+involvement+of+COX-2+and+apoptosis-related+proteins%5BTitle%5D)+AND+%22J.+Physiol.+Pharmacol%22%5BJournal%5D)">Inhibition of Barret's adenocarcinoma cell growth by simvastatin: involvement of COX-2 and apoptosis-related proteins.</a> J. Physiol. Pharmacol. 58 Suppl 3, 141-148 (2007).</li>
<li>Peng, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glutathione+peroxidase+7+protects+against+oxidative+DNA+damage+in+oesophageal+cells%5BTitle%5D)+AND+%22Gut%22%5BJournal%5D)">Glutathione peroxidase 7 protects against oxidative DNA damage in oesophageal cells.</a> Gut. 61, 1250-1260 (2012).</li>
<li>Vona-Davis, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MAPK+and+PI3K+inhibition+reduces+proliferation+of+Barrett%27s+adenocarcinoma+in+vitro%5BTitle%5D)+AND+%22Surg.+Res%22%5BJournal%5D)">MAPK and PI3K inhibition reduces proliferation of Barrett's adenocarcinoma in vitro.</a> Surg. Res. 127, 53-58 (2005).</li>
<li>Inge, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((a+small+molecule+inhibitor+of+the+Src+kinase%2C+reduces+the+growth+and+activates+apoptosis+in+pre-neoplastic+Barrett%27s+esophagus+cell+lines%3A+evidence+for+a+noninvasive+treatment+of+high-grade+dysplasia%5BTitle%5D)+AND+%22Thoracic+Cardiovasc.+Surg%22%5BJournal%5D)">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/pubmed?term=((Ion+transport+and+barrier+function+in+a+telomerase-immortalized+human+nondysplastic%2C+Barrett%27s+cell+line+%28BAR-T%29%5BTitle%5D)+AND+%22Dis.+Esophagus%22%5BJournal%5D)">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/pubmed?term=((An+in+vitro+co-culture+model+of+esophageal+cells+identifies+ascorbic+acid+as+a+modulator+of+cell+competition%5BTitle%5D)+AND+%22BMC+Cancer%22%5BJournal%5D)">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/pubmed?term=((In+benign+Barrett%27s+epithelial+cells%2C+acid+exposure+generates+reactive+oxygen+species+that+cause+DNA+double-strand+breaks%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">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/pubmed?term=((Extended+lifespan+of+Barrett%27s+esophagus+epithelium+transduced+with+the+human+telomerase+catalytic+subunit%3A+a+useful+in+vitro+model%5BTitle%5D)+AND+%22Carcinogenesis%22%5BJournal%5D)">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/pubmed?term=((Src+activation+in+malignant+and+premalignant+epithelia+of+Barrett%27s+esophagus%5BTitle%5D)+AND+%22Gastroenterology%22%5BJournal%5D)">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/pubmed?term=((alpha-Catenin+overrides+Src-dependent+activation+of+beta-catenin+oncogenic+signaling%5BTitle%5D)+AND+%22Mol.+Cancer+Ther%22%5BJournal%5D)">alpha-Catenin overrides Src-dependent activation of beta-catenin oncogenic signaling.</a> Mol. Cancer Ther. 7, 1386-1397 (2008).</li>
<li>Coluccia, A. M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SKI-606+Decreases+Growth+and+Motility+of+Colorectal+Cancer+Cells+by+Preventing+pp60%28c-Src%29-Dependent+Tyrosine+Phosphorylation+of+%CE%B2-Catenin+and+Its+Nuclear+Signaling%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">SKI-606 Decreases Growth and Motility of Colorectal Cancer Cells by Preventing pp60(c-Src)-Dependent Tyrosine Phosphorylation of β-Catenin and Its Nuclear Signaling.</a> Cancer Res. 66, 2279-2286 (2006).</li>
<li>Nam, J. -S., Ino, Y., Sakamoto, M., Hirohashi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Src+Family+Kinase+Inhibitor+PP2+Restores+the+E-Cadherin%2FCatenin+Cell+Adhesion+System+in+Human+Cancer+Cells+and+Reduces+Cancer+Metastasis%5BTitle%5D)+AND+%22Clin.+Cancer+Res%22%5BJournal%5D)">Src Family Kinase Inhibitor PP2 Restores the E-Cadherin/Catenin Cell Adhesion System in Human Cancer Cells and Reduces Cancer Metastasis.</a> Clin. Cancer Res. 8, 2430-2436 (2002).</li>
<li>Chu, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((p27+phosphorylation+by+Src+regulates+inhibition+of+cyclin+E-Cdk2%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">p27 phosphorylation by Src regulates inhibition of cyclin E-Cdk2.</a> Cell. 128, 281-294 (2007).</li>
<li>Inge, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Soluble+E-cadherin+promotes+cell+survival+by+activating+epidermal+growth+factor+receptor%5BTitle%5D)+AND+%22Exp.+Res%22%5BJournal%5D)">Soluble E-cadherin promotes cell survival by activating epidermal growth factor receptor.</a> Exp. Res. 317, 838-848 (2011).</li>
<li>Gan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+roles+of+ERK+and+Akt+pathways+in+regulation+of+EGFR-mediated+signaling+and+motility+in+prostate+cancer+cells%5BTitle%5D)+AND+%22Oncogene%22%5BJournal%5D)">Differential roles of ERK and Akt pathways in regulation of EGFR-mediated signaling and motility in prostate cancer cells.</a> Oncogene. 29, 4947-4958 (2010).</li>
</ol>

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 choice of fixation. We have described the use of 4% PFA/PBS for fixation in the above procedures but alternative fixatives are also applicable. Fixation with cold methanol (-20&#160;&#176;C) for 20 min after washing with PBS is suitable for certain proteins/antibodies13. Use of cold methanol for fixation negates the need to permeabilize cells. However, the choice of fixative (4% PFA/PBS or cold methanol) must be determined empirically.
The procedures outlined above describe visualization of a single protein; however this method is easily adaptable to identification of an additional target13. For optimal IF of two proteins, primary antibodies raised in two different species (e.g.,&#160;one raised in rabbit and one in mouse), and secondary antibodies specifically recognizing each species, coupled to different fluorophores, are necessary. Secondary antibodies coupled to green and red fluorophores, combined with DAPI provide an excellent contrast between targets, in addition to generally avoiding interference from the multiple fluorophores. For IF of two targets in BE cells, perform sequential incubation of two primary and their respective secondary antibodies following fixation. Steps for washing and mounting of slides remain the same.
In summary, we provide a simple and useful protocol for IF of BE cells. Application of these procedures can yield information regarding the effects of drug treatment, introduction of ectopic genes of interest, or RNAi downregulation of genes of interest in BE cells.

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><tbody><tr><td>CP-A (Metaplastic Cell Line)</td><td>ATCC</td><td>CRL-4027</td><td></td></tr><tr><td>CP-B (High-grade Dysplastic Cell Line)</td><td>ATCC</td><td>CRL-4028</td><td></td></tr><tr><td>CP-C (High-grade Dysplastic Cell Line)</td><td>ATCC</td><td>CRL-4029</td><td></td></tr><tr><td>CP-D (High-grade Dysplastic Cell Line)</td><td>ATCC</td><td>CRL-4030</td><td></td></tr><tr><td>Keratinocyte-SFM (1x), Liquid</td><td>Life Technologies</td><td>17005-042</td><td></td></tr><tr><td>0.25% Trypsin-EDTA (1x), Phenol Red&amp;nbsp;</td><td>Life Technologies</td><td>25200056</td><td></td></tr><tr><td>Fetal Bovine Serum, Qualified, HI</td><td>Life Technologies</td><td>10438026</td><td></td></tr><tr><td>PSN antibiotic mixture</td><td>Life Technologies</td><td>15640</td><td></td></tr><tr><td>PBS - Phosphate-Buffered Saline</td><td>Life Technologies</td><td>10010049</td><td></td></tr><tr><td>Pro-long Gold Antifade Reagent with DAPI&amp;nbsp;</td><td>Life Technologies</td><td>P36931</td><td></td></tr><tr><td>Circular Glass Coverslip 18 mm</td><td>Fisher Scientific</td><td>15-183-86</td><td></td></tr><tr><td>&amp;beta;-catenin Primary Antibody (Rabbit)</td><td>Cell Signaling</td><td>9562S</td><td></td></tr><tr><td>Alexa Fluor 488 (Goat Anti-Rabbit)</td><td>Life Technologies</td><td>A11008</td><td></td></tr><tr><td>10 cm TC treated PS dish, sterile</td><td>USA Scientific</td><td>CC7682-3394</td><td></td></tr><tr><td>12-well TC treated PS plate, sterile</td><td>USA Scientific</td><td>5666-5180</td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>276855</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td></td></tr><tr><td>Ammonium chloride</td><td>Sigma-Aldrich</td><td>A9434</td><td></td></tr><tr><td>Saponin</td><td>Sigma-Aldrich</td><td>47036</td><td></td></tr><tr><td>Bovine Serum Albumin - Fraction V</td><td>Sigma-Aldrich</td><td>85040C</td><td></td></tr><tr><td>SKI-606 (Bosutinib)</td><td>Selleck Chemicals</td><td>S1014</td><td></td></tr><tr><td>Square Bioassay Dish&amp;nbsp;</td><td>Thermo Scientific</td><td>240835</td><td></td></tr><tr><td>Parafilm&amp;nbsp;</td><td>VWR</td><td>82024-546</td><td></td></tr><tr><td>Disposable Pasteur Pipettes, Flint Glass</td><td>VWR</td><td>14672-380</td><td></td></tr><tr><td>Nexcelom Mini Cell Counter</td><td>Nexcelom</td><td></td><td></td></tr><tr><td>Cellometer Counting Chambers</td><td>Nexcelom</td><td>CHT4-SD100-014</td><td></td></tr><tr><td>Zeiss Apotome microscope&amp;nbsp;</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 accomplished by labeling with DAPI (blue). Coverslips were mounted to microscope slides using hard set mounting and cells were imaged using laser scanning confocal microscope using a 63X/1.4N plan-apochromat objective. The chosen green fluorophore was excited at 488 nm and visualized using an emission filter at 505-525 nm. Similarly, DAPI was excited at 405 nm and emission collected using a filter at 410-460 nm. Activity of the tyrosine kinase, Src is increased in BE, particularly in high grade dysplasia12. In concordance with observations in colorectal and prostate cancer cells13-15, inhibition of Src results in relocalization of &#946;-catenin from the cytoplasm/nucleus (arrows, Figures 1A, 1C, and 1E) to the cell membrane (arrowheads, Figures 1B, 1D, and 1F). Using these techniques, we and others have previously shown that inhibition of Src also alters the expression and localization of the tumor suppressor, p27kip17,16 and these methods are currently used within our laboratory.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51741/51741fig1highres.jpg" width="500" />&#956; 
Figure 1. IF of immortalized BE cells following treatment with an inhibitor of Src kinase. CP-C (A-D) and CP-D (E, F) were treated with vehicle (DMSO, left panels) or 1 &#956;M of the Src inhibitor, SKI-606, for 24 hr (right panels). BE cells were fixed and stained according to the described protocol. &#946;-catenin (green) was visualized using a specific antibody and a secondary antibody coupled to Alexa Fluro 488, while the nucleus was stained with DAPI (blue). Note the change in localization for &#946;-catenin from the nucleus (arrows) to the cell membrane (arrowheads) following treatment with SKI-606. C,D) Magnified images of white boxes in A and B, further highlight the changes in &#946;-catenin localization. These images represent ideal staining with minimal background and distinct staining for the chosen target. Bars, 7 &#956;m (A, B, D, F), 0.5 &#956;m (C, D).

Watch this Scientific Journal Video about An Immunofluorescent Method for Characterization of BarrettÃ¢â‚¬â„¢s Esophagus Cells at JoVE.com

An Immunofluorescent Method for Characterization of Barrett&#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.

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 ...

an-immunofluorescent-method-for-characterization-barretts-esophagus

Nanyang Technological University

Research

JoVE Journal

Biology

14.0K Views.  St. Joseph's Hospital and Medical Center.  Er is duidelijk een behoefte aan betere informatie over de moleculaire bestuurders van Barrett-slokdarm. Immunofluorescentie kleuring is een nuttige techniek voor het begrijpen van de effecten van celsignalen op celmorfologie. Wij presenteren een eenvoudig, efficiënt protocol voor het gebruik van immunofluorescentie kleuring therapeutische behandeling van Barrett-slokdarm cellen bepalen. 

Video: An Immunofluorescent Method for Characterization of Barrett’s Esophagus Cells

Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinoma Samples

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their relative distribution and their localization in the tissue. This technique has been extensively applied to study the immune infiltrate in various tumor types. The tumor microenvironment is infiltrated by immune cells that are attracted to the tumor site. Different immune cell populations have been found to play different roles in the tumor microenvironment and to have a different impact on the outcome of disease. This manuscript describes the use of multiparameter fluorescence IHC on oropharyngeal squamous cell carcinoma (OPSCC) as an example. This technique can be extended to other tissue samples and cell types of interest. In the presented study, we analyzed the intraepithelial and stromal compartment of a large OPSCC cohort (n = 162). We focused on total T lymphocytes (CD3+), immunosuppressive regulatory T cells (Tregs; i.e., FoxP3+), and T helper 17 (Th17) cells (i.e., IL-17+CD3+) using a nuclear counterstain to distinguish tumor epithelium from stroma. A high number of T cells was found to be correlated with improved disease-free survival in patients with a low number of intratumoral IL-17+ non-T cells. This suggests that IL-17+ non-T cells may be correlated with a poor immune response in OPSCC, which is in agreement with the correlation described between IL-17 and poor survival in cancer patients. Currently, novel multiparameter fluorescence IHC techniques are being developed using up to 7 different fluorochromes and will enable the more precise characterization and localization of immune cells in the tumor microenvironment.

Multiparameter fluorescence immunohistochemistry can be used to assess the number, relative distribution, and localization of immune cell populations in the tumor microenvironment. This manuscript describes the use of this technique to analyze T-cell subpopulations in oropharyngeal cancer.

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their ...

Cancer Research

Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority of CRC cases, cell dysfunction is the result of numerous mutations that modify protein expression and post-translational modification1.
A number of cell surface antigens, including cluster of differentiation (CD) antigens, have been identified as potential prognostic or metastatic biomarkers in CRC. These antigens make ideal biomarkers as their expression often changes with tumour progression or interactions with other cell types, such as tumour-infiltrating lymphocytes (TILs) and tumour-associated macrophages (TAMs).
The use of immunohistochemistry (IHC) for cancer sub-classification and prognostication is well established for some tumour types2,3. However, no single &#x2018;marker&#x2019; has shown prognostic significance greater than clinico-pathological staging or gained wide acceptance for use in routine pathology reporting of all CRC cases. 
A more recent approach to prognostic stratification of disease phenotypes relies on surface protein profiles using multiple 'markers'. While expression profiling of tumours using proteomic techniques such as iTRAQ is a powerful tool for the discovery of biomarkers4, it is not optimal for routine use in diagnostic laboratories and cannot distinguish different cell types in a mixed population. In addition, large amounts of tumour tissue are required for the profiling of purified plasma membrane glycoproteins by these methods. 
In this video we described a simple method for surface proteome profiling of viable cells from disaggregated CRC samples using a DotScan CRC antibody microarray. The 122-antibody microarray consists of a standard 82-antibody region recognizing a range of lineage-specific leukocyte markers, adhesion molecules, receptors and markers of inflammation and immune response5, together with a satellite region for detection of 40 potentially prognostic markers for CRC. Cells are captured only on antibodies for which they express the corresponding antigen. The cell density per dot, determined by optical scanning, reflects the proportion of cells expressing that antigen, the level of expression of the antigen and affinity of the antibody6. 
For CRC tissue or normal intestinal mucosa, optical scans reflect the immunophenotype of mixed populations of cells. Fluorescence multiplexing can then be used to profile selected sub-populations of cells of interest captured on the array. For example, Alexa 647-anti-epithelial cell adhesion molecule (EpCAM; CD326), is a pan-epithelial differentiation antigen that was used to detect CRC cells and also epithelial cells of normal intestinal mucosa, while Phycoerythrin-anti-CD3, was used to detect infiltrating T-cells7. The DotScan CRC microarray should be the prototype for a diagnostic alternative to the anatomically-based CRC staging system.

We described a procedure for the disaggregation of colorectal cancer (CRC) to produce viable single cells, which are then captured on customized antibody microarrays recognizing surface antigens (DotScan CRC microarray). Sub-populations of cells bound to the microarray can be profiled by fluorescence multiplexing using monoclonal antibodies tagged with fluorescent dyes.

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority ...

Immunology and Infection

Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens

Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of tissue samples from formalin-fixed, paraffin-embedded (FFPE) biopsies has become a key tool for understanding the complexity of tumor immunology and discovering novel predictive biomarkers for cancer immunotherapy. Immunoprofiling analysis of tissues requires the evaluation of combined markers, including inflammatory cell subpopulations and immune checkpoints, in the tumor microenvironment. The advent of novel multiplex immunohistochemical methods allows for a more efficient multiparametric analysis of single tissue sections than does standard monoplex immunohistochemistry (IHC). One commercially available multiplex immunofluorescence (IF) method is based on tyramide-signal amplification and, combined with multispectral microscopic analysis, allows for a better signal separation of diverse markers in tissue. This methodology is compatible with the use of unconjugated primary antibodies that have been optimized for standard IHC on FFPE tissue samples. Herein we describe in detail an automated protocol that allows multiplex IF labeling of carcinoma tissue samples with a six-marker multiplex antibody panel comprising PD-L1, PD-1, CD68, CD8, Ki-67, and AE1/AE3 cytokeratins with 4&#8242;,6-diamidino-2-phenylindole as a nuclear cell counterstain. The multiplex panel protocol is optimized in an automated IHC stainer for a staining time that is shorter than that of the manual protocol and can be directly applied and adapted by any laboratory investigator for immuno-oncology studies on human FFPE tissue samples. Also described are several controls and tools, including a drop-control method for fine quality control of a new multiplex IF panel, that are useful for the optimization and validation of the technique.

A detailed protocol for a six-marker multiplex immunofluorescence panel is optimized and performed, using an automated stainer for more consistent results and a shorter procedure time. This approach can be directly adapted by any laboratory for immuno-oncology studies.

Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of ...

Diagnosis of Neoplasia in Barrett&#8217;s Esophagus using Vital-dye Enhanced Fluorescence Imaging

The ability to differentiate benign metaplasia in Barrett&#8217;s Esophagus (BE) from neoplasia in vivo remains difficult as both tissue types can be flat and indistinguishable with white light imaging alone. As a result, a modality that highlights glandular architecture would be useful to discriminate neoplasia from benign epithelium in the distal esophagus. VFI is a novel technique that uses an exogenous topical fluorescent contrast agent to delineate high grade dysplasia and cancer from benign epithelium. Specifically, the fluorescent images provide spatial resolution of 50 to 100 &#956;m&#160;and a field of view up to 2.5 cm,&#160;allowing endoscopists to visualize glandular morphology. Upon excitation, classic Barrett&#8217;s metaplasia appears as continuous, evenly-spaced glands and an overall homogenous morphology; in contrast, neoplastic tissue appears crowded with complete obliteration of the glandular framework. Here we provide an overview of the instrumentation and enumerate the protocol of this new technique. While VFI affords a gastroenterologist with the glandular architecture of suspicious tissue, cellular dysplasia cannot be resolved with this modality. As such, one cannot morphologically distinguish Barrett&#8217;s metaplasia from BE with Low-Grade Dysplasia via this imaging modality. By trading off a decrease in resolution with a greater field of view, this imaging system can be used at the very least as a red-flag imaging device to target and biopsy suspicious lesions; yet, if the accuracy measures are promising, VFI may become the standard imaging technique for the diagnosis of neoplasia (defined as either high grade dysplasia or cancer) in the distal esophagus.

Vital-dye enhanced fluorescence imaging (VFI) is a novel in vivo technique that combines high-resolution epithelial imaging with exogenous topical fluorescent contrast to highlight glandular morphology and delineate neoplasia (high grade dysplasia and cancer) in the distal esophagus.

The ability to differentiate benign metaplasia in Barrett&#8217;s Esophagus (BE) from neoplasia in vivo remains difficult as both tissue types can be flat ...

Bioengineering

Multiplexed Barcoding Image Analysis for Immunoprofiling and Spatial Mapping Characterization in the Single-Cell Analysis of Paraffin Tissue Samples

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is an effective methodology for tumor evaluation that improves the understanding of the tumor microenvironment. The visualization of protein expression in formalin-fixed, paraffin-embedded&#160;tissues is achieved when a specific fluorophore is annealed to an antibody-bound barcode via complementary oligonucleotides and then sample imaging is performed; indeed, this method allows for the use of customizable panels of more than 40 antibodies in a single tissue staining reaction. This method is compatible with fresh frozen tissue, formalin-fixed, paraffin-embedded tissue, cultured cells, and peripheral blood mononuclear cells, meaning that researchers can use this technology to view a variety of sample types at single-cell resolution. This method starts with a manual staining and fixing protocol, and all the antibody barcodes are applied using an antibody cocktail. The staining fluidics instrument is fully automated and performs iterative cycles of labeling, imaging, and removing spectrally distinct fluorophores until all the biomarkers have been imaged using a standard fluorescence microscope. The images are then collected and compiled across all the imaging cycles to achieve single-cell resolution for all the markers. The single-step staining and gentle fluorophore removal not only allow for highly multiplexed biomarker analysis but also preserve the sample for additional downstream analysis if desired (e.g., hematoxylin and eosin staining). Furthermore, the image analysis software enables image processing-drift compensation, background subtraction, cell segmentation, and clustering-as well as the visualization and analysis of the images and cell phenotypes for the generation of spatial network maps. In summary, this technology employs a computerized microfluidics system and fluorescence microscope to iteratively hybridize, image, and strip fluorescently labeled DNA probes that are complementary to tissue-bound, oligonucleotide-conjugated antibodies.

Multiplexed barcoding image analysis has recently improved the characterization of the tumor microenvironment, permitting comprehensive studies of cell composition, functional state, and cell-cell interactions. Herein, we describe a staining and imaging protocol using the barcoding of oligonucleotide-conjugated antibodies and cycle imaging, which allows for the use of a high-dimensional image&#160;analysis technique.

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is ...

Human Esophageal Organoids and Air-Liquid Interface Culture Protocols

Three-Dimensional Cell Culture Models to Investigate the Epithelial Barrier in Eosinophilic Esophagitis

The squamous epithelium of the esophagus is directly exposed to the environment, continuously facing foreign antigens, including food antigens and microbes. Maintaining the integrity of the epithelial barrier is critical for preventing infections and avoiding inflammation caused by harmless food-derived antigens. This article provides simplified protocols for generating human esophageal organoids and air-liquid interface cultures from patient biopsies to study the epithelial compartment of the esophagus in the context of tissue homeostasis and disease. These protocols have been significant scientific milestones in the last decade, describing three-dimensional organ-like structures from patient-derived primary cells, organoids, and air-liquid interface cultures. They offer the possibility to investigate the function of specific cytokines, growth factors, and signaling pathways in the esophageal epithelium within a three-dimensional framework while maintaining the phenotypic and genetic properties of the donor. Organoids provide information on tissue microarchitecture by assessing the transcriptome and proteome after cytokine stimulation. In contrast, air-liquid interface cultures allow the assessment of the epithelial barrier integrity through transepithelial resistance (TEER) or macromolecule flux measurements. Combining these organoids and air-liquid interface cultures is a powerful tool to advance research in impaired esophageal epithelial barrier conditions.

Author Spotlight: Investigating the Pathophysiology of Eosinophilic Esophagitis

Here, a protocol for the culture of human esophageal organoids and air-liquid interface culture is provided. Esophageal organoids' air-liquid interface culture can be used to study the impact of cytokines on the esophageal epithelial barrier.

The squamous epithelium of the esophagus is directly exposed to the environment, continuously facing foreign antigens, including food antigens and ...

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, ...

Functional Characterization and Visualization of Esophageal Fibroblasts Using Organoid Co-Cultures

Epithelial stem and progenitor cells contribute to the formation and maintenance of the epithelial barrier throughout life. Most stem and progenitor cell populations are tucked away in anatomically distinct locations, enabling exclusive interactions with niche signals that maintain stemness. While the development of epithelial organoid cultures provides a powerful tool for understanding the role of stem and progenitor cells in homeostasis and disease, the interaction within the niche environment is largely absent, thereby hindering the identification of factors influencing stem cell behavior. Fibroblasts play a key role in directing epithelial stem and progenitor fate. Here, a comprehensive organoid-fibroblast co-culture protocol enabling the delineation of fibroblast subpopulations in esophageal progenitor cell renewal and differentiation is presented. In this protocol, a method to isolate both epithelial cells and fibroblasts in parallel from the esophagus is described. Distinct fluorescence-activated cell sorting strategies to isolate both the esophageal progenitor cells as well as the fibroblast subpopulations from either transgenic reporter or wild-type mice are outlined. This protocol provides a versatile approach that can be adapted to accommodate the isolation of specific fibroblast subpopulations. Establishing and passaging esophageal epithelial organoid mono-cultures is included in this protocol, enabling a direct comparison with the co-culture system. In addition, a 3D clearing approach allowing for detailed image analysis of epithelial-fibroblast interactions is described. Collectively, this protocol describes a comparative and relatively high-throughput method for identifying and understanding esophageal stem cell niche components in vitro.

Organoid-fibroblast co-cultures provide a model to study the in vivo stem cell niche. Here, a protocol for esophageal organoid-fibroblast co-cultures is described. Additionally, whole mount imaging is used to visualize the fibroblast-organoid interaction.

Epithelial stem and progenitor cells contribute to the formation and maintenance of the epithelial barrier throughout life. Most stem and progenitor cell ...

CTC Immunofluorescence Assay : A Technique to  Characterize Circulating Tumor Cells

Source: Garcia, J., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/59873/semi-automatic-pd-l1-characterization-enumeration-circulating-tumor">Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence.</a>&#160;J. Vis. Exp.&#160;(2019)
This video describes the Circulating Tumor Cells (CTC) Immunofluorescence assay, a technique to characterize the biomarkers on circulating tumor cells in non-small cell lung cancer patient samples. This assay could be used to monitor dynamic changes of the tumor and immune response, which may reflect the response to chemotherapy, radiation, and possible immunotherapy treatments.

Source: Garcia, J., et al. Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by ...

JoVE Encyclopedia of Experiments

Lung Cancer

Ex vivo study

Multiplexed Immunofluorescence Imaging to Analyze Immunotherapy-Resistant T Cell Subpopulations

Source: Granier, C., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56606">Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells.</a> J. Vis. Exp. (2018).
This video demonstrates an in-situ fluorescence multispectral imaging technique to analyze different subpopulations of infiltrating T cells in tumor tissue. A renal cell carcinoma tissue cryosection is treated with antibodies to label CD8+ T cells that express specific inhibitory receptors and exhibit an immunotherapy-resistant phenotype. The labeled section is visualized using a multispectral microscope to identify the distinct CD8+ T cell subpopulations.

An Immunofluorescent Method for Characterization of Barrett’s Esophagus Cells

Samenvatting

Meer video's verkennen