This work was supported by National Institutes of Health Grants R01DK120986 (to K.P.M.).

Epithelial Effects of Intestinal Inflammation on Established Murine Colonoids

<ol>
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The contributing authors have nothing to disclose.

Organoid development has revolutionized the way the scientific community studies organ systems in vitro with the ability to partially recapitulate cellular structure and function from an animal or human in a dish. Further, organoid systems derived from humans with diseases offer a promising tool for personalized medicine that could guide therapeutic decision-making. Here, we&#160;describes a crypt isolation protocol that works well and introduces key steps that allow for cleaning up excess debris in the isolatio...

Inflammatory bowel disease (IBD) is a chronic, remitting, and relapsing disease characterized by episodes of inflammation and clinical quiescence. The etiology of IBD is multifactorial, but key characteristic features of the disease include defective barrier function and increased permeability of the intestinal epithelium, in addition to proinflammatory signaling cascades activated within the epithelial compartment1,2. Several in vitro and in vivo models have been used to recapitulate the epithelial response during IBD, including cell culture and murine models of inflammation3. However, all these systems have important shortcomings that limit their ability to recapitulate the epithelial changes during IBD4. Most cell lines used to study IBD are transformed, have the ability to form a monolayer, and can differentiate3 but intrinsically propagate differently than non-transformed intestinal epithelial cells in the host. Several different murine models of inflammation are used to study IBD, some of which include knockout models, infectious models, chemical inflammatory models, and T-cell transfer models5,6,7,8. While each can study certain etiological aspects of IBD, such as genetic predispositions, barrier dysfunction, immune deregulation, and the microbiome, they are limited in their ability to study the multifactorial nature of the disease.
Intestinal organoids, including enteroids and colonoids, have been established over the last decade as a useful in vitro model for studying not only the dynamics of intestinal stem cells but also their role the barrier integrity and function of the intestinal epithelium play in intestinal homeostasis and disease. These entities have significantly contributed to our understanding of the pathogenesis of IBD9 and have opened new opportunities for personalized medicine. Colonoids, or stem cell-derived, self-organizing tissue cultures from the colon, have been developed from both murine and human tissue in a process that allows stem cells located within intestinal crypts to propagate and be maintained indefinitely10. The stem cell niche in vivo relies on extracellular factors to support its growth, notably the canonical Wnt signaling and bone-morphogenic protein signaling pathways11. The addition of these factors promotes the health and longevity of colonoids but also drives the culture toward a stem cell-like state that is not reflective of the in vivo epithelial cellular architecture, which consists of both self-renewing and terminally differentiated cells12,13. While the functionality of the intestinal epithelium is dependent upon the continual crosstalk between the stem cell compartment and differentiated cells, the ability to have both in a colonoid culture system is fairly limited. Despite these limitations, the organoid culture system remains the gold standard to study the intrinsic properties of the epithelium ex vivo. Nonetheless, alternative culture strategies may need to be considered to answer the scientific question at hand.
It has been shown that mice on a continuous 7 day regimen of dextran sodium sulfate (DSS) develop both epithelial inflammation and barrier dysfunction14. Furthermore, mitochondrial biogenesis failure and metabolic reprogramming within the intestinal epithelium, which have been shown to be evident in human IBD, have also been captured in this DSS model of colitis15. However, our preliminary data demonstrate that the characteristics of mitochondrial biogenesis failure are not retained in colonoids derived from the crypts of DSS-treated animals (Supplementary Figure 1). Thus, alternative culture methods must be used when examining how inflammation drives epithelial changes during murine intestinal inflammation. Here, we outline a protocol we have developed that describes 1) how to isolate crypts from whole colonic tissue for the establishment of murine colonoids, 2) how to terminally differentiate this cell population to reflect the cell population as it stands in vivo, and 3) how to induce inflammation in this in vitro model. To study drug interactions within the inflamed epithelium, we have developed a protocol to establish 2-dimensonal (2D) monolayers from murine colonoids that can be basally treated with inflammatory mediators and apically treated with drug therapies.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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		<tr>
			<td>0.4-&#956;M transparent transwell, 24-well</td>
			<td>Greiner Bio-one</td>
			<td>662-641</td>
			<td></td>
		</tr>
		<tr>
			<td>15-mL conical tubes</td>
			<td>Thermo Fisher&#160;</td>
			<td>12-565-269</td>
			<td></td>
		</tr>
		<tr>
			<td>50-mL conical tubes</td>
			<td>Thermo Fisher&#160;</td>
			<td>12-565-271</td>
			<td></td>
		</tr>
		<tr>
			<td>70-&#956;M cell strainer</td>
			<td>VWR</td>
			<td>76327-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Invitrogen</td>
			<td>12634-010</td>
			<td>Stock Concentration (1x); Final Concentration (1x)</td>
		</tr>
		<tr>
			<td>B-27 supplement&#160;</td>
			<td>Invitrogen</td>
			<td>12587-010</td>
			<td>Stock Concentration (50x); Final Concentration (1x)</td>
		</tr>
		<tr>
			<td>Chopsticks Electrode Set for EVO</td>
			<td>World Precision Instruments</td>
			<td>STX2</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Matrigel GFR Membrane Mix</td>
			<td>Corning</td>
			<td>354-230</td>
			<td>Stock Concentration (100%); Final Concentration (100%)</td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Sigma-Aldrich</td>
			<td>D0632-5G</td>
			<td>Stock Concentration (1 M); Final Concentration (1.5 mM); Solvent (ultrapure water)</td>
		</tr>
		<tr>
			<td>DMEM high glucose</td>
			<td>Thermo Fisher</td>
			<td>11960-069</td>
			<td>Stock Concentration (1x); Final Concentration (1x)</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline without Calcium and Magnesium</td>
			<td>Gibco&#160;</td>
			<td>14190-144</td>
			<td>Stock Concentration (1x); Final Concentration (1x)</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (ETDA)</td>
			<td>Sigma-Aldrich</td>
			<td>E7889</td>
			<td>Stock Concentration (0.5 M); Final Concentration (30 mM)</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Bio-Techne</td>
			<td>S11150H</td>
			<td>Stock Concentration (100%); Final Concentration (1%)</td>
		</tr>
		<tr>
			<td>Fisherbrand Superfrost Plus Microscope Slides, White, 25 x 75 mm</td>
			<td>Thermo Fisher&#160;</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>G418</td>
			<td>InvivoGen</td>
			<td>ant-ga-1</td>
			<td>Final Concentration (400 &#181;g/&#181;L)</td>
		</tr>
		<tr>
			<td>Gentamicin Reagent</td>
			<td>Gibco/Fisher</td>
			<td>15750-060</td>
			<td>Stock Concentration (50 mg/mL); Final Concentration (250 &#956;g/mL)</td>
		</tr>
		<tr>
			<td>GlutaMAX-1</td>
			<td>Fisher Scientific</td>
			<td>35050-061</td>
			<td>Stock Concentration (100x); Final Concentration (1x)</td>
		</tr>
		<tr>
			<td>HEPES 1 M</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td>Stock Concentration (1 M); Final Concentration (10 mM)</td>
		</tr>
		<tr>
			<td>hIFN&#947;</td>
			<td>R&#38;D Systems</td>
			<td>285-IF</td>
			<td>Stock Concentration (1000 ng/&#181;L); Final Concentration (10 ng/mL); Solvent (ultrapure water)</td>
		</tr>
		<tr>
			<td>hIL-1&#946;</td>
			<td>R&#38;D Systems</td>
			<td>201-LB</td>
			<td>Stock Concentration (10 ng/&#181;L); Final Concentration (20 ng/mL); Solvent (ultrapure water)</td>
		</tr>
		<tr>
			<td>hTNF&#945;</td>
			<td>R&#38;D Systems</td>
			<td>210-TA</td>
			<td>Stock Concentration (10 ng/&#181;L); Final Concentration (40 ng/mL); Solvent (ultrapure water)</td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide&#160;</td>
			<td>Sigma</td>
			<td>H1009</td>
			<td>Stock Concentration (30%); Final Concentration (0.003%); Solvent (Mouse wash media)</td>
		</tr>
		<tr>
			<td>Hygromycin B Gold</td>
			<td>InvivoGen</td>
			<td>ant-hg-1</td>
			<td>Final Concentration (400 &#181;g/&#181;L)</td>
		</tr>
		<tr>
			<td>L-WRN Cell Line</td>
			<td>ATCC</td>
			<td>CRL-3276</td>
			<td></td>
		</tr>
		<tr>
			<td>mEGF</td>
			<td>Novus</td>
			<td>NBP2-35176</td>
			<td>Stock Concentration (0.5 &#181;g/&#181;L); Final Concentration (50 ng/mL); Solvent (D-PBS + 1% BSA)</td>
		</tr>
		<tr>
			<td>N-2 supplement</td>
			<td>Invitrogen</td>
			<td>17502-048</td>
			<td>Stock Concentration (100x); Final Concentration (1x)</td>
		</tr>
		<tr>
			<td>N-Acetyl-L-cysteine</td>
			<td>Sigma&#160;</td>
			<td>A9165-5G</td>
			<td>Stock Concentration (500 mM); Final Concentration (1 mM); Solvent (ultrapure water)</td>
		</tr>
		<tr>
			<td>Noggin</td>
			<td>Peprotech</td>
			<td>250-38</td>
			<td>Stock Concentration (0.1 ng/&#181;L); Final Concentration (100 ng/mL); Solvent (UltraPure water + 0.1% BSA)</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermo Fisher</td>
			<td>15140-122</td>
			<td>Stock Concentration (100x); Final Concentration (1x)</td>
		</tr>
		<tr>
			<td>Petri dishes (sterilized; 100 mm x 15 mm) Polystrene disposable</td>
			<td>VWR</td>
			<td>25384-342</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene Microplates, 24 well tissue culture treated, sterile</td>
			<td>Greiner Bio-one</td>
			<td>5666-2160</td>
			<td></td>
		</tr>
		<tr>
			<td>R-Spondin</td>
			<td>R&#38;D Systems</td>
			<td>3474-RS-050</td>
			<td>Stock Concentration (0.25 &#181;g/&#181;L); Final Concentration (500 ng/mL); Solvent (D-PBS + 1% BSA)</td>
		</tr>
		<tr>
			<td>Tryp LE Express</td>
			<td>Thermo Fisher</td>
			<td>12604-013</td>
			<td>Stock Concentration (10x); Final Concentration (1x); Solvent (1 mM EDTA)</td>
		</tr>
		<tr>
			<td>UltraPure Water&#160;</td>
			<td>Invitrogen</td>
			<td>10977-023</td>
			<td>Stock Concentration (1x); Final Concentration (1x)</td>
		</tr>
		<tr>
			<td>Y-27632 dihyddrochloride&#160;</td>
			<td>Abcam</td>
			<td>ab120129</td>
			<td>Stock Concentration (10 mM); Final Concentration (10 &#181;M); Solvent (UltraPure Water)</td>
		</tr>
	</tbody>
</table>

studying the epithelial effects of intestinal inflammation in vitro on established murine colonoids

The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic cell layer provides the first line of defense between microbial and immune populations and helps to modulate the intestinal immune response. Disruption of the epithelial barrier is a hallmark of inflammatory bowel disease (IBD) and is of interest for therapeutic targeting. The 3-dimensional colonoid culture system is an extremely useful in vitro model for studying intestinal stem cell dynamics and epithelial cell physiology in IBD pathogenesis. Ideally, establishing colonoids from the inflamed epithelial tissue of animals would be most beneficial in assessing the genetic and molecular influences on disease. However, we have shown that in vivo epithelial changes are not necessarily retained in colonoids established from mice with acute inflammation. To address this limitation, we have developed a protocol to treat colonoids with a cocktail of inflammatory mediators that are typically elevated during IBD. While this system can be applied ubiquitously to various culture conditions, this protocol emphasizes treatment on both differentiated colonoids and 2-dimensional monolayers derived from established colonoids. In a traditional culture setting, colonoids are enriched with intestinal stem cells, providing an ideal environment to study the stem cell niche. However, this system does not allow for an analysis of the features of intestinal physiology, such as barrier function. Further, traditional colonoids do not offer the opportunity to study the cellular response of terminally differentiated epithelial cells to proinflammatory stimuli. The methods presented here provide an alternative experimental framework to address these limitations. The 2-dimensional monolayer culture system also offers an opportunity for therapeutic drug screening ex vivo. This polarized layer of cells can be treated with inflammatory mediators on the basal side of the cell and concomitantly with putative therapeutics apically to determine their utility in IBD treatment.

The 3D intestinal colonoid culture system is an invaluable tool to study the intrinsic contribution of the epithelium to intestinal mucosal homeostasis. The described protocol provides detailed instructions on how to isolate crypts from C57BL/6J (WT) mice at 8 weeks of age and establish a long-term colonoid culture system that can be manipulated for multiple downstream applications. Upon the isolation and plating of crypts in the basement membrane matrix, the crypts appear dense and multicellular in structure when visual...

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Author Spotlight: Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids  

We describe a protocol detailing the isolation of murine colonic crypts for the development of 3-dimensional colonoids. The established colonoids can then be terminally differentiated to reflect the cellular composition of the host epithelium prior to receiving an inflammatory challenge or being directed to establish an epithelial monolayer.

Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids

The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic ...

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899 Views.  UPMC Children’s Hospital of Pittsburgh. We describe a protocol detailing the isolation of murine colonic crypts for the development of 3-dimensional colonoids. The established colonoids can then be terminally differentiated to reflect the cellular composition of the host epithelium prior to receiving an inflammatory challenge or being directed to establish an epithelial monolayer.

Video: Author Spotlight: Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids  

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and cardiovascular disease1-11. Traditional cell biological approaches to studying cellular function involve culturing cells on a rigid substratum (plastic dishes or glass coverslips) which cannot account for the effect of an elastic ECM or the variations in ECM stiffness between tissues. To model in vivo tissue compliance conditions in vitro, we and others use ECM-coated hydrogels. In our laboratory, the hydrogels are based on polyacrylamide which can mimic the range of tissue compliances seen biologically12. "Reactive" cover slips are generated by incubation with NaOH followed by addition of 3-APTMS. Glutaraldehyde is used to cross-link the 3-APTMS and the polyacrylamide gel. A solution of acrylamide (AC), bis-acrylamide (Bis-AC) and ammonium persulfate is used for the polymerization of the hydrogel. N-hydroxysuccinimide (NHS) is incorporated into the AC solution to crosslink ECM protein to the hydrogel. Following polymerization of the hydrogel, the gel surface is coated with an ECM protein of choice such as fibronectin, vitronectin, collagen, etc.
The stiffness of a hydrogel can be determined by rheology or atomic force microscopy (AFM) and adjusted by varying the percentage of AC and/or bis-AC in the solution12. In this manner, substratum stiffness can be matched to the stiffness of biological tissues which can also be quantified using rheology or AFM. Cells can then be seeded on these hydrogels and cultured based upon the experimental conditions required. Imaging of the cells and their recovery for molecular analysis is straightforward. For this article, we define soft substrata as those having elastic moduli (E) &lt;3000 Pascal and stiff substrata/tissues as those with E &gt;20,000 Pascal.

The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and ...

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Investigating Intestinal Inflammation in DSS-induced Model of IBD

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease (CD). Both UC and CD, when present in the colon, generate a similar symptom profile which can include diarrhea, rectal bleeding, abdominal pain, and weight loss.1 Although the pathogenesis of IBD remains unknown, it is described as a multifactorial disease that involves both genetic and environmental components.2
There are numerous and variable animal models of colonic inflammation that resemble several features of IBD. Animal models of colitis range from those arising spontaneously in susceptible strains of certain species to those requiring administration of specific concentrations of colitis-inducing chemicals, such as dextran sulphate sodium (DSS). Chemical-induced models of gut inflammation are the most commonly used and best described models of IBD. Administration of DSS in drinking water produces acute or chronic colitis depending on the administration protocol.3 Animals given DSS exhibit weight loss and signs of loose stool or diarrhea, sometimes with evidence of rectal bleeding.4,5 Here, we describe the methods by which colitis development and the resulting inflammatory response can be characterized following administration of DSS. These methods include histological analysis of hematoxylin/eosin stained colon sections, measurement of pro-inflammatory cytokines, and determination of myeloperoxidase (MPO) activity, which can be used as a surrogate marker of inflammation.6
The extent of the inflammatory response in disease state can be assessed by the presence of clinical symptoms or by alteration in histology in mucosal tissue. Colonic histological damage is assessed by using a scoring system that considers loss of crypt architecture, inflammatory cell infiltration, muscle thickening, goblet cell depletion, and crypt abscess.7 Quantitatively, levels of pro-inflammatory cytokines with acute inflammatory properties, such as interleukin (IL)-1&beta;, IL-6 and tumour necrosis factor (TNF)-&alpha;,can be determined using conventional ELISA methods. In addition, MPO activity can be measured using a colorimetric assay and used as an index of inflammation.8
In experimental colitis, disease severity is often correlated with an increase in MPO activity and higher levels of pro-inflammatory cytokines. Colitis severity and inflammation-associated damage can be assessed by examining stool consistency and bleeding, in addition to assessing the histopathological state of the intestine using hematoxylin/eosin stained colonic tissue sections. Colonic tissue fragments can be used to determine MPO activity and cytokine production. Taken together, these measures can be used to evaluate the intestinal inflammatory response in animal models of experimental colitis.

Experimental models of inflammatory bowel disease have allowed us to examine the complex innate and adaptive immune responses associated with pathogenesis. Using histological scoring, quantification of pro-inflammatory cytokines and myeloperoxidase activity, one can begin to assess these responses seen in inflammatory bowel disease.

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Biochemical Titration of Glycogen In vitro

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2) versus hypoxia (1% O2). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive1.

Biochemical Titration of Glycogen In vitro

We describe here an accurate, reproducible and convenient biochemical method for titration of glycogen in vitro. This technique uses the Abcam Glycogen assay kit and is based on successive hydrolysis of glycogen to glucose and glucose titration by fluorescence.

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

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

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

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

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.