This work was supported by a Grant-in-Aid for Scienti&#64257;c Research C (grant no. 19K09834 to K.N.) from the Ministry of Education, Science, and Culture, Japan.

All procedures were performed under the protocol approved by the Ethics Committee for human welfare. All patients provided written informed consent for the research use of their samples, and the collection and use of tissues for this study were approved by the Human Genome, Gene Analysis Research Ethics Committee at Teikyo University.
1. Isolation and culture of ovarian cancer stem cells (OCSCs) from Patients with ovarian cancer and ascites in a level 2 biosafety cabinet
<ol>
	<li>Isolate cancer stem cells from human ovarian cancer ascites obtained via paracentesis. Collect at least 100-250 mL of ascites from patients to take enough number of cancer stem cells. Additionally, evaluate the expression profiles of cancer stem cell markers (i.e., EpCAM, Calretinin, CD133, CD44, CD45, ALDH1, and Oct4) and ovarian cancer markers (pAX-8, WT-1) by flow cytometry.
	<ol>
		<li>Centrifuge the human ovarian cancer ascites at 300 x g for 10 min at room temperature within 24 h after ascites aspiration.</li>
		<li>Remove the supernatant and add 2 mL of OCSC medium and 8 mL of 30% Histodenz/phosphate buffered saline (PBS, pH 7.4) solution.</li>
		<li>Prepare OCSC medium: StemPro hESC supplement, DMEM&#8260;F-12 with L-glutamine (GlutaMAX medium), 25% BSA, 100 &#181;M 2-mercaptoethanol, 8 ng/mL FGF BASIC, 10 &#181;M insulin, and 20 &#181;M Y-27632.</li>
		<li>Carefully overlay 2 mL of OCSC medium to the cell solution in step 1.1.2 in a 15 mL tube and centrifuge at 450 x g for 20 min at room temperature in a swinging-bucket rotor without braking.</li>
		<li>Carefully transfer the OCSC layer (undisturbed at the interphase) to a new 15 mL tube by transfer pipet.</li>
		<li>Fill with PBS up to 15 mL. Centrifuge at 300 x g for 5 min at room temperature and remove the supernatant.</li>
		<li>Resuspend the cell pellet in OCSC medium and seed on an ultra-low-attachment culture dish; cultures should be maintained at 37 &#176;C in 5% CO2.</li>
		<li>Change the medium every three days. Carefully stand the culture dish for about 1 minutes, and discard part of the supernatant and add the new medium.</li>
	</ol>
	</li>
	<li>Passage of CSCs
	<ol>
		<li>Collect OCSCs in a 15 mL tube and centrifuge at 200 x g for 5 min at room temperature.</li>
		<li>Remove the supernatant, fill with PBS, and centrifuge at 200 x g for 5 min at room temperature.</li>
		<li>Remove the supernatant, add 1 mL of the cell detachment solution consisting of proteolytic and collagenolytic enzymes (e.g., AccuMax), and incubate at 37 &#176;C for 10 min.</li>
		<li>Mix well by pipetting and incubate at 37 &#176;C for 5 min. Ensure cells are in a single suspension.</li>
		<li>Mix well by pipetting, fill with PBS, and centrifuge at 300 x g for 5 min at room temperature.</li>
		<li>Remove the supernatant and resuspend the cell pellet in OCSC medium for subsequent seeding on ultra-low-attachment culture dishes and maintenance at 37 &#176;C in 5% CO2.</li>
	</ol>
	</li>
</ol>
2. HUEhT-1 endothelial cell culture
<ol>
	<li>Passage of HUEhT-1 cells
	<ol>
		<li>Remove medium from the HUEhT-1 culture dish and wash the cells with PBS.</li>
		<li>Add 1 mL of 0.025% trypsin and incubate for 3 min at room temperature.</li>
		<li>Add 5 mL of Endothelial Cell Growth Medium 2, collect cells in a 15 mL tube, and centrifuge at 200 x g for 5 min at room temperature.</li>
		<li>Remove the supernatant, resuspend the cell pellet in HUEhT-1 medium, and seed the cells on collagen-coated culture dishes followed by maintenance at 37 &#176;C in 5% CO2.</li>
		<li>Change the medium every three days.</li>
	</ol>
	</li>
</ol>
3. Preparation of the NICO-1 Coculture Plate for tube formation assay using HUEhT-1 cells
<ol>
	<li>Assemble NICO-1 and coating with the extracellular matrix-based hydrogel (Matrigel Matrix).
	<ol>
		<li>Assemble one side of NICO-1 following the manufacturer&#39;s instructions and keep on ice.</li>
		<li>Cover the surface of NICO-1 with 300 &#956;L of cold PBS and then remove the buffer.</li>
		<li>Add 300 &#956;L of chilled extracellular matrix-based hydrogel and incubate at 37 &#176;C for 60 min.</li>
		<li>To equilibrate, immerse the filter with 100% ethanol, then wash a 13 mm ICCP Filter (0.6 &#181;m) with PBS for 1 min.</li>
		<li>Assemble the NICO-1 including both main body parts A (right chamber) and B (left chamber) along with the O ring and equilibrated filter.</li>
	</ol>
	</li>
</ol>
4. Seeding HUEhT-1 Cells and CSCs onto the NICO-1 system
<ol>
	<li>Prepare HUEhT-1 cell suspensions by trypsinizing the cell monolayers and resuspending the cells in endothelial cell growth medium with 2% fetal calf serum.</li>
	<li>Add 1.2 mL of the cell suspension (1.5 x 105 cells) to each extracellular matrix-based hydrogel-coated well.</li>
	<li>Add 1.5 mL of OCSCs cultured for five days to the other well.</li>
	<li>Incubate NICO-1 at 37 &#176;C in 5% CO2; tube formation can be observed under the microscope and network formation on extracellular matrix-based hydrogel measured by means of the number of branches.</li>
</ol>

<ol>
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The authors have nothing to disclose.

The presented protocol describes how to mimic the tumor microenvironment of OCSCs in an in vitro setting. The primary component of the method constitutes the highly reproducible coculture model obtained using the NICO-1 system, an indirect Transwell co-culture system. Many of the currently available coculture models examine the effects of direct cell-cell contact on cocultured cell populations12,13,14,<sup class="xref...

Ovarian cancer is the eighth most common malignancy in women worldwide, with approximately 300,000 new diagnoses and an estimated 180,000 deaths annually1. At initial diagnosis, ovarian cancer often presents with severe symptoms, with about 75% of patients already at stage III-IV. Accordingly, the 5-year survival rate is &#60;30% and the mortality rate is the highest among gynecological cancers2, with the efficiency of treatment for ovarian cancer being highly dependent on clinical factors such as the successful accomplishment of debulking surgery, resistance to chemotherapy, and recurrence after the initial therapy.
Ovarian cancer tissues are hierarchically organized, with not all tumor components being equally capable of generating descendants. The only cells able to self-renew and produce a heterogeneous tumor cell population are considered to represent cancer stem cells (CSCs)3. CSC self-renewal and tumor initiation are accompanied by the promotion of angiogenesis to remodel their tumor microenvironment for the purpose of maintaining a supportive niche. However, previous models could not be utilized for in vitro analyses because of the limited reproducibility of cultivating CSCs derived from clinical samples owing to the disruption of spheroids after multiple passaging. More recently, experimental methods to cultivate CSCs from patients have been developed for several applications4,5,6,7. In particular, by exploiting the characteristic of CSCs to grow by forming spheroids in ultra-low attachment plates with serum-free medium, the cultivated CSCs are induced to express a stem-cell surface marker that is not expressed in normal tumor cells with multilineage differentiation potential8,9.
Recent data have shown that the persistence of dormant ovarian (O)CSCs visualized as dissemination at the peritoneum is associated with their regeneration as recurrent tumors10. Understanding the molecular and biological features of OCSCs may thus allow for effective targeting and eradication of these cells, resulting in potential tumor remission. In particular, little is known regarding the cellular and molecular mechanistic features of CSCs roles in angiogenesis11. Therefore, in the present protocol we used patient-derived OCSCs in an in vitro setting to investigate the angiogenic property of endothelial cells using the co-culture model, which may mimic the tumor microenvironment of CSCs and endothelial cells at the metastatic site in the clinical setting. Ultimately, as neovascularization constitutes a critical process necessary to support tumor growth and metastasis, a better understanding of its mechanism will allow the development of a novel targeting therapy for OCSCs at the metastatic site.
Here, we present a protocol to visualize the neovascularization step surrounding the CSCs in a time course manner. The advantage of the protocol includes allowing fully reproducible investigations using the 3D co-culture system, NICO-1, thereby permitting observation of the effects on patients of the OCSC-derived tumor-initiation capability during endothelial cell angiogenesis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.025% Trypsin&#160;</td>
			<td>Thermo</td>
			<td>R001100</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Pipet</td>
			<td>Thermo</td>
			<td>170356N</td>
			<td></td>
		</tr>
		<tr>
			<td>1250 &#181;L Pipet tip</td>
			<td>QSP</td>
			<td>T112XLRS-Q</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL tube</td>
			<td>Nunc</td>
			<td>339650</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;L Pipet tip</td>
			<td>QSP</td>
			<td>T110RS-NEW</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Thermo (Gibco)</td>
			<td>21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Pipet</td>
			<td>Thermo</td>
			<td>170366N</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL tube</td>
			<td>Corning</td>
			<td>430290</td>
			<td></td>
		</tr>
		<tr>
			<td>AccuMAX</td>
			<td>Innovative Cell Technologies</td>
			<td>AM105</td>
			<td></td>
		</tr>
		<tr>
			<td>BioCoatTM Collagen I 60mm Dish</td>
			<td>Corning</td>
			<td>356401</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>KUBOTA</td>
			<td>2800</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar 6 Well Clear Flat Bottom Ultra Low Attachment Multiple Well Plates</td>
			<td>Corning</td>
			<td>3471</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth Medium 2&#160;</td>
			<td>PromoCell</td>
			<td>C-22011&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>WAKO</td>
			<td>057-00456</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF-Basic</td>
			<td>Thermo (Gibco)</td>
			<td>PHG0021</td>
			<td></td>
		</tr>
		<tr>
			<td>Histodenz</td>
			<td>SIGMA</td>
			<td>D2158</td>
			<td></td>
		</tr>
		<tr>
			<td>HUEhT-1 cell</td>
			<td>JCRB Cell Bank</td>
			<td>JCRB1458</td>
			<td></td>
		</tr>
		<tr>
			<td>ICCP Filter 0.6 &#181;m</td>
			<td>Ginrei Lab.</td>
			<td>2525-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin, human</td>
			<td>SIGMA (Roche)</td>
			<td>11376497001</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminometer</td>
			<td>PerkinElmer</td>
			<td>ARVO MX-flad</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Matrix</td>
			<td>Corning</td>
			<td>356234</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Yokogawa</td>
			<td>CQ-1</td>
			<td></td>
		</tr>
		<tr>
			<td>NICO-1</td>
			<td>Ginrei Lab.</td>
			<td>2501-02</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiPlate-96</td>
			<td>PerkinElmer</td>
			<td>6005290</td>
			<td></td>
		</tr>
		<tr>
			<td>P1000 Pipet</td>
			<td>Gilson</td>
			<td>F123602</td>
			<td></td>
		</tr>
		<tr>
			<td>P200 Pipet</td>
			<td>Gilson</td>
			<td>F123601</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Thermo (Gibco)</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro hESC SFM</td>
			<td>Thermo (Gibco)</td>
			<td>A1000701</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer Pipet</td>
			<td>FALCON</td>
			<td>357575</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>WAKO</td>
			<td>253-00513</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluating the angiogenetic properties of ovarian cancer stem-like cells using the three-dimensional co-culture system, nico-1

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular matrix. The ability of CSCs to participate in the development of endothelium constitutes an important characteristic that directly contributes to the general understanding of the mechanisms of tumorigenesis and tumor metastasis. The purpose of this work is to establish a reproducible methodology to investigate the tumor-initiation capability of ovarian cancer stem cells (OCSCs). Herein, we examined the neovascularization mechanism between endothelial cells and OCSCs along with the morphological changes of endothelial cells using the in vitro co-culture model NICO-1. This protocol allows visualization of the neovascularization step surrounding the OCSCs in a time course manner. The technique can provide insight regarding the angiogenetic properties of OCSCs in tumor metastasis.

We collected ascites fluids obtained from patients with advanced ovarian cancer during surgery or paracentesis for the purpose of performing a long-term stable culture for spheroids. Here, we present cases of a long-term spheroid culture of ovarian CSCs termed CSC1 and CSC2. Both cell lines carry the same diagnosis and histological profiles. The mechanistic roles of OCSCs underlying the interaction with endothelial cells necessary to induce the neovascularization of endothelial cells surr...

Watch this Scientific Journal Video about Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-Like Cells using the Three-Dimensional Co-Culture System, NICO-1 at JoVE.com

Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-Like Cells using the Three-Dimensional Co-Culture System, NICO-1

Ovarian cancer stem cells (OCSC) are responsible for cancer initiation, recurrence, therapeutic resistance, and metastasis. The OCSC vascular niche is considered to promote self-renewal of OCSCs, leading to chemoresistance. This protocol provides the basis for establishing a reproducible OCSC vascular niche model in vitro.

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular ...

evaluating-angiogenetic-properties-ovarian-cancer-stem-like-cells

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2.5K Views.  Teikyo University School of Medicine. Ovarian cancer stem cells (OCSC) are responsible for cancer initiation, recurrence, therapeutic resistance, and metastasis. The OCSC vascular niche is considered to promote self-renewal of OCSCs, leading to chemoresistance. This protocol provides the basis for establishing a reproducible OCSC vascular niche model in vitro.

Video: Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-Like Cells using the Three-Dimensional Co-Culture System, NICO-1

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being used to evaluate the structure and function of plant xylem network in three dimensions (3D) (e.g. Brodersen et al. 2010; 2011; 2012a,b). HRCT imaging is based on the same principles as medical CT systems, but a high intensity synchrotron x-ray source results in higher spatial resolution and decreased image acquisition time. Here, we demonstrate in detail how synchrotron-based HRCT (performed at the Advanced Light Source-LBNL Berkeley, CA, USA) in combination with Avizo software (VSG Inc., Burlington, MA, USA) is being used to explore plant xylem in excised tissue and living plants. This new imaging tool allows users to move beyond traditional static, 2D light or electron micrographs and study samples using virtual serial sections in any plane. An infinite number of slices in any orientation can be made on the same sample, a feature that is physically impossible using traditional microscopy methods.
Results demonstrate that HRCT can be applied to both herbaceous and woody plant species, and a range of plant organs (i.e. leaves, petioles, stems, trunks, roots). Figures presented here help demonstrate both a range of representative plant vascular anatomy and the type of detail extracted from HRCT datasets, including scans for coast redwood (Sequoia sempervirens), walnut (Juglans spp.), oak (Quercus spp.), and maple (Acer spp.) tree saplings to sunflowers (Helianthus annuus), grapevines (Vitis spp.), and ferns (Pteridium aquilinum and Woodwardia fimbriata). Excised and dried samples from woody species are easiest to scan and typically yield the best images. However, recent improvements (i.e. more rapid scans and sample stabilization) have made it possible to use this visualization technique on green tissues (e.g. petioles) and in living plants. On occasion some shrinkage of hydrated green plant tissues will cause images to blur and methods to avoid these issues are described. These recent advances with HRCT provide promising new insights into plant vascular function.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...

Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine system, but the complex structural relationships and three-dimensional (3D) vasculature architectures of the ovary are not well described. In order to visualize the 3D connections and architecture of blood vessels in the intact ovary, the first important step is to make the ovary optically clear. In order to avoid tissue shrinkage, we used the hydrogel fixation-based passive CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/ Immunostaining/In situ-hybridization-compatible Tissue Hydrogel) protocol method to clear an intact ovary. Immunostaining, advanced multiphoton confocal microscopy, and 3D image-reconstructions were then used for the visualization of ovarian vessels and follicular capillaries. Using this approach, we showed a significant positive correlation (P &#60;0.01) between the length of the follicular capillaries and volume of the follicular wall.

Here we present an adaptation of the passive CLARITY and 3D reconstruction method for visualization of the ovarian vasculature and follicular capillaries in intact mouse ovaries.

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine ...

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

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

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

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

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development ...