Name: primary endodermal epithelial cell culture from the yolk sac membrane of japanese quail embryos
Uploaded: 2016-03-10T13:00:00
Description: Watch this Scientific Journal Video about Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos at JoVE.com

Funding for the development of current protocol is particularly supported by &#8220;Aim for the Top University Plan&#8221; of the National Taiwan University, Taiwan (grant ID-104R350144), as well as the Ministry of Science and Technology of Taiwan (grant ID: MOST 104-2313-B-002-039-MY3). We especially thank the Center for Biotechnology of National Taiwan University for providing an animal room and laboratory space for the current study.

NOTE: This procedure is a modification of a chicken model culture protocol developed by Bauer et al. 2013 and Nakazawa et al., 2011. 2,9
1. Prepare Healthy Embryonic Day 5 Embryos from Japanese Quail
<ol>
	<li>Place 1 male and 3 female sexually mature Japanese quail together in the same cage. Supply feed and water ad libitum. Adjust the light in the animal room to 14 hr of light and 10 hr of darkness.</li>
	<li>Collect fertilized eggs in plastic bags eve...

<ol>
	<li>Abeyrathne, E. D., Lee, H. Y., Ahn, D. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Egg+white+proteins+and+their+potential+use+in+food+processing+or+as+nutraceutical+and+pharmaceutical+agents--a+review.">Egg white proteins and their potential use in food processing or as nutraceutical and pharmaceutical agents--a review.</a> Poult Sci. 92 (12), 3292-3299 (2013).</li><li>Bauer, R., Plieschnig, J. A., Finkes, T., Riegler, B., Hermann, M., Schneider, W. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transport+across+endodermal+cells+of+the+chick+yolk+sac+during+early+stages+of+development.">Transport across endodermal cells of the chick yolk sac during early stages of development.</a> Am J Anat. 160 (3), 285-308 (1981).</li><li>Nakazawa, F., Alev, C., Jakt, L. M., Sheng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yolk+sac+endoderm+is+the+major+source+of+serum+proteins+and+lipids+and+is+involved+in+the+regulation+of+vascular+integrity+in+early+chick+development.">Yolk sac endoderm is the major source of serum proteins and lipids and is involved in the regulation of vascular integrity in early chick development.</a> Dev Dyn. 240 (8), 2002-2010 (2011).</li><li>Noble, R. C., Cocchi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+metabolism+and+the+neonatal+chicken.">Lipid metabolism and the neonatal chicken.</a> Prog Lipid Res. 29 (2), 107-140 (1990).</li><li>Shand, J. H., West, D. W., McCartney, R. J., Noble, R. C., Speake, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+esterification+of+cholesterol+in+the+yolk+sac+membrane+of+the+chick+embryo.">The esterification of cholesterol in the yolk sac membrane of the chick embryo.</a> Lipids. 28 (7), 621-625 (1993).</li><li>Speier, J. S., Yadgary, L., Uni, Z., Wong, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+of+nutrient+transporters+and+digestive+enzymes+in+the+yolk+sac+membrane+and+small+intestine+of+the+developing+embryonic+chick.">Gene expression of nutrient transporters and digestive enzymes in the yolk sac membrane and small intestine of the developing embryonic chick.</a> Poult Sci. 91 (8), 1941-1949 (2012).</li><li>Walzem, R. L., Hansen, R. J., Williams, D. L., Hamilton, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+Induction+of+VLDLy+Assembly+in+Egg-Laying+Hens.">Estrogen Induction of VLDLy Assembly in Egg-Laying Hens.</a> J Nutr. 129 (2), 467S-472S (1999).</li><li>Yoshizaki, N., Soga, M., Ito, Y., Mao, K. 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Because the previous culture system has only limited success, a better culture system is needed. The Japanese quail YSM endoderm requires treatment with proteolytic enzymes such as collagenase to loosen the cell-cell junctions to achieve better growth performance in the ex-vivo explants. Our data show that cell numbers from partial digestion treatment were greater than from the undigested tissue culture after seeding for 2 days (Figure 1). Therefore, the partial proteolytic digestion is a critical step t...

The major nutritional resource of the avian embryo is yolk, composed of 33% lipids, 17% protein, and 1% ash.1 During embryonic development, the yolk sac membrane (YSM) grows from within the embryonic abdominal cavity and gradually covers the yolk surface. Beginning at embryonic day 2, gene expression associated with lipid metabolism and angiogenesis is gradually increased in YSM, and the YSM slowly develops villus-like projections.8,9 These projections increase absorption of yolk nutrients to support embryonic development. The YSM is an extraembryonic tissue that contains three germ layers, endoderm, mesoderm and ectoderm.14 The yolk sac ectoderm faces the albumen and links with the vitelline membrane to gently cover the yolk sac. The endodermal epithelial cells are faced directly toward the egg yolk and serve as nutrient utilization portals.6 As the YSM expands, endodermal epithelial cell (EECs) can be divided by shape and functionality into two groups, area vitelline and area vasculosa.7
Area vitelline is composed of endodermal cells and is distant from the embryo; area vasculosa is composed of mesodermal cells and covers differentiated EECs with blood vessels and connective tissues. By embryonic day 5, the yolk is totally covered by ectoderm and endoderm of YSM and the vascular area has grown rapidly. The YSM absorbs, recomposes and releases lipids (as yolk-derived very low density lipoprotein) and proteins into the embryonic circulatory system.9, 2 Therefore, we established a primary Japanese quail embryonic endodermal epithelial cell culture system, to study the mechanisms of lipid utilization in YSM during avian embryonic development.
Lipids such as triacylglycerol, lecithin, phospholipid and cholesterol ester (CE) are the primary energy sources for avian embryos. At the early stages of development, yolk lipids are composed of only 1.3% CE and it rises to 10-15% at mid-term of avian embryonic development 3, 11. Cholesterol ester is synthesized from cholesterol by sterol O-acyltransferase 1 (SOAT1) in avian embryo YSM.4
The storage form of cholesterol is CE, CE is carried in lipoproteins, and lipoproteins are transported by circulation to tissues.13 A week before hatch there is rapid growth of avian embryos. Approximately 68% of the remaining lipid contents in yolk are absorbed during this stage.10 The mechanism by which yolk lipids are utilized can be clarified by an EEC research model. A chicken EEC culture protocol was established to achieve this research goal.2, 9 However, due to the low success rate of tissue explants, an improved EEC cell culture procedure is needed to study the function of certain enzymes and proteins in mediating nutrient utilization by avian embryos during development.

<table border="0" cellpadding="0" cellspacing="0" width="845">
	<tbody>
		<tr height="50">
			<td height="50" style="height:50px;width:331px;">Dulbecco&#39;s Modified Eagle Medium</td>
			<td style="width:199px;">Gibco by Life technologies</td>
			<td style="width:148px;">12800-017 10 X 1 L</td>
			<td style="width:167px;">For wash the EECs pellets</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:331px;">D-MEM/F-12</td>
			<td style="width:199px;">Gibco by Life technologies</td>
			<td style="width:148px;">12400-024 10 X 1 L</td>
			<td>As the basal medium in culturing EECs</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:331px;">NBCS</td>
			<td style="width:199px;">Gibco by Life technologies</td>
			<td style="width:148px;">16010-159</td>
			<td>As the supplyment serum in culturing EECs</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:331px;">Pen-Strep Ampho. Solution</td>
			<td style="width:199px;">BI (Biological Industries)</td>
			<td style="width:148px;">03-033-1B 100ml</td>
			<td>For attenuating the possible infection&#160;</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:331px;">Collagenase Type IV</td>
			<td style="width:199px;">Gibco by Life technologies</td>
			<td style="width:148px;">17104-019 1g</td>
			<td>&#160;Collagenase is a protease with specificity for the bond between a neutral amino acid (X) and glycine in the sequence Pro-XGly-Pro. As the protease for dissociation of cells from primary tissue.</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:331px;">24 well plate</td>
			<td style="width:199px;">FALCON&#174;</td>
			<td style="width:148px;">REF-353047</td>
			<td>For EECs to attach and extension</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:331px;">50 ML PP centrifuge tubes</td>
			<td style="width:199px;">Corning&#174; CentriStarTM</td>
			<td style="width:148px;">430829</td>
			<td>For transportion of membranes and enzyme digestion</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">50ML Conical bottomed Tube with Cap</td>
			<td style="width:199px;">PRO TECH</td>
			<td>CT-50-PL-TW</td>
			<td>For transportion of membranes and enzyme digestion</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:331px;">Reciprocal shaking bath</td>
			<td>DEAGLE</td>
			<td style="width:148px;">SB302</td>
			<td>For better enzymatic digestion on membranes</td>
		</tr>
	</tbody>
</table>

primary endodermal epithelial cell culture from the yolk sac membrane of japanese quail embryos

We established an endodermal epithelial cell culture model (EEC) for studying the function of certain enzymes and proteins in mediating nutrient utilization by avian embryos during development. Fertilized Japanese quail eggs were incubated at 37 &#176;C for 5 days and then yolk sac membranes (YSM) were collected to establish the EEC culture system. We isolated the embryonic endoderm layer from YSM, and sliced the membrane into 2 - 3 mm pieces and partially digested with collagenase before seeding in 24-well culture plates. The EECs proliferate out of the tissue and are ready for cell culture studies. We found that the EECs had typical characteristics of YSM in vivo, for example, accumulation of lipid droplets, expression of sterol O-acyltransferase and lipoprotein lipase. The partial digestion treatment significantly increased the successful rate of EEC culture. Utilizing the EECs, we demonstrated that the expression of SOAT1 was regulated by the cAMP dependent protein kinase A related pathway. This primary Japanese quail EEC culture system is a useful tool to study embryonic lipid transportation and to clarify the role of genes involved in mediating nutrient utilization in YSM during avian embryonic development.

In order to achieve the goal of establishing a consistent and useful cell model, we need to extend and stabilize the proliferation rate and performance of avian EECs. We compared direct incubation of the endoderm with no enzyme digestion with endoderm partially digested with proteolytic enzymes, such as collagenase or collagenase plus 0.6 U of dispase. Dispase is an amino-endopeptidase that hydrolyzes the N-terminal peptide bonds of non-polar amino acid residues. Whereas protease digestio...

Watch this Scientific Journal Video about Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos at JoVE.com

Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos

To study the mechanism of lipid utilization in yolk sac membranes during the late stages of avian embryonic development, we established a primary Japanese quail embryonic endodermal epithelial cell culture system.

We established an endodermal epithelial cell culture model (EEC) for studying the function of certain enzymes and proteins in mediating nutrient ...

The overall goal of this ex vivo explant procedure is to improve the collection and extend the application of avian endodermal epithelial cells in order to study the function of enzymes and proteins in mediating nutrient utilization by avian embryos. This method can help answer key questions regarding lipid utilization during avian embryogenesis, such as how the yolk lipids are packaged and transported from the yolk to the embryo during the later stages of development. The main advantage of this technique is that we can successfully explain functional endodermal epithelial cells from Japanese quail yolk sac membrane, which can be utilized as a model to study avian embryonic development.Begin by making a 10x PBS solution as described in the text protocol. Dilute this stock solution one to ten to prepare 1x PBS, which will be used to wash the yolk sac membrane during endoderm dissection. To prepare growth medium for endodermal epithelial cells, or EECs, first supplement DMEM/F-12 with 10%newborn calf serum and 1%penicillin streptomycin amphotericin solution.Afterwards, insert the probe of a pH meter into previously prepared DMEM. Add three normal HCl dropwise to the medium until the pH has been adjusted to 7.2. After collecting approximately two dozen fertilized quail eggs, place them vertically in egg containers so that their wide, untapered ends, where the air cells are located, are pointing upward.Then, transfer the eggs to a well-ventilated incubator set at 37 degrees Celsius. After five days of incubation, examine the eggs with an egg candler. If embryos have developed normally, expect to see a circular structure of capillary vessels beneath the air cell.Select an egg demonstrating normal capillary development, and clean the shell with fresh water and 75%ethanol. Next, open the egg shell from the air cell position using scissors. Use forceps to gently help pour out the contents of the egg into an empty 10 cm culture dish.Then, use scissors to detach the embryo, yolk, and any albumin from the yolk sac membrane, which is abbreviated as YSM. Once isolated, wash the YSM three times in 1x PBS, and then, transfer it to a new 10 cm dish containing PBS. Afterwards, place the dish under a dissecting scope.Then, proceed to remove any residual egg white, which abuts the outer ectoderm layer of the YSM, with scissors. Continue by using forceps to lift the translucent ectoderm and red capillary structural mesoderm from the edge of the mesoderm on the yolk sac membrane. Then, gently peel the ectoderm and mesoderm together away to isolate the underlying endoderm cell layer.Then, with one pair of forceps, firmly hold the yellow endodermal cell layer of the membrane. With another pair, grip the capillary mesoderm, and proceed to pull the mesoderm away from the junction edge of the mesoderm and endoderm. Using a pair of scissors, cut off the remaining yellow endoderm layer, and transfer it to the six centimeter culture dish filled with growth medium.Using a dropper, transfer the light yellow endoderm from the six centimeter culture dish to a 15 mL centrifuge tube after YSM collection, and to it, add 15 mL of previously prepared growth medium. Place the conical in a 37 degrees Celsius water bath until all tissue collections are completed. Begin by dissolving 6.5 units of Type IV collagenase in 10 mL of DMEM.Next, centrifuge the endoderm-containing tubes at 130 time g for three minutes at room temperature. Following centrifugation, aspirate the medium, and use a dropper to transfer all the endoderm samples to a six centimeter culture dish. Then, add one milliliter of collagen solution to the dish.With curved scissors, proceed to cut the endoderm collected from four quail embryos into slices approximately two to three millimeters in size. Afterwards, collect the slices into a 50 mL centrifuge tube, and with nine milliliters of fresh collagenase solution, which is already inside of the 50 mL centrifuge tube. To improve cell proliferation during the explant stage, place the tube in a shaking water bath set to 37 degrees Celsius.Then, incubate the conical for 30 minutes to partially digest the tissue with collagenase. The partial proteolytic digestion is the most important step to improve the cell yolk from yolk sac membrane endoderm collection. Our data shows a significant improvement of the successful rate of cell culture and cell proliferation by the collagenase digestion procedure.Following collagenase digestion, centrifuge the tissue at 130 times g for three minutes at room temperature, and then remove the supernatant fraction with a pipette. Proceed to wash the pellet by resuspending it in 20 mL of DMEM. Centrifuge the tube as previously described, then afterwards, remove the supernatant, and resuspend the pellet into 12 mL of growth medium.Then, gently add 500 microliters of the suspension to each well of a 24-well plate. Proceed to incubate the endoderm explants for two days at 37 degrees Celsius in 5%CO2. During this time, expect cells to proliferate out of the tissues.Next, incubate the cells for another two days. Then, subject them to a cell viability assay, and visualize the results using a plate reader. Afterwards, perform RT-qPCR to evaluate expression levels of markers of interest in EECs and check the product size from RT-qPCR by gel electrophoresis as described in the text protocol.Compared to untreated explants, those partially digested with collagenase exhibited significantly increased cell proliferation, as shown in these graphs and determined by a paired t-test, demonstrating the success of the culture system. To evaluate the effects of other enzymes, cell viability assays were performed for tissues treated with collagenase and dispase or with collagenase alone. Over the first five days of incubation, cell growth was similar in both conditions.However, from days six to nine, only collagenase-treated EECs proliferated steadily, suggesting that partial digestion with this enzyme improves growth and should be preferentially used. When cultured EECs were visualized, they demonstrated the adipocyte-like features of large circular lipid droplets as shown here. Such cells were also positive for Oil Red O staining, which is typically used to identify adipocytes.Supporting these morphological data, real-time quantitative PCR revealed that cultured EECs demonstrate high levels of SOAT1, a protein that synthesizes cholesterol ester from cholesterol. This further suggests that cultured EECs demonstrate the correct physiological characteristics of adipocytes. Importantly, when EECs were exposed to IBMX and Forskolin, factors that increase cyclic adenosine monophosphate, or cAMP levels, and cultured for 24 hours, both treatments stimulated SOAT1 expression, suggesting possible mechanisms of SOAT1 regulation and lipid utilization in the YSM.Once mastered, this technique can be done in about four hours, during which time, at least three samples can be collected. Following this procedure, methods like SOAT1 or other enzymatic activity assays as well as similar visual confirmation can be performed. Such assay may help answer additional questions, like how certain protein and macronutrients are involved in the later stages of embryonic development.After its development, this technique paved the way for researchers in the field of embryonic nutrient utilization to explore the possible mechanism by which nutrients are utilized and transported in an avian embryonic model. After watching this video, you should be able to learn how to collect endoderm tissues from avian embryos, and also know how to culture EECs for studying embryonic nutrient transportation and utilization using the avian model.

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Developmental Biology

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The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex&#160;ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols1. In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Embryonic chick retinal cell cultures constitute valuable tools for the study of photoreceptor biology. We have developed an efficient gene transfer technique based on ex&#160;ovo plasmid electroporation of the retinas prior to culture. This technique considerably increases transfection efficiencies over currently available protocols, making genetic manipulation feasible for this system.

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

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The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish embryos has been implemented. This technique takes advantage of the selectivity and sensitivity conferred by affinity labeling of a given receptor by its ligand with the specificity of the immunoprecipitation. We have used AFLIP to detect the type III TGF-&#946; receptor (TGFBR3), also know as betaglycan, during early zebrafish development. AFLIP was instrumental in validating the efficacy of a TGFBR3 morphant zebrafish phenotype. In the first step, embryo protein extracts are prepared and used to generate 125I-TGF-&#946;2-TGFBR3 complexes that are purified by immunoprecipitation. Later, these complexes are covalently cross-linked and revealed using SDS-PAGE separation and autoradiography detection. This technique requires the availability of a labeled ligand for, and a specific antibody against, the receptor to be detected, and shall be easily adapted to identify any growth factor or cytokine receptor that meets these requirements.

A novel technique for the detection of low abundance endogenous receptors present in zebrafish embryos is described. We have named it AFLIP because it consists of affinity labeling of the receptor by its ligand linked to immunoprecipitation.

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Whole-cell Patch-clamp Recordings of Isolated Primary Epithelial Cells from the Epididymis

The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types that are distinct not only in molecular and morphological features but also in physiological properties. These differences reflect their diverse functions, which together establish the necessary microenvironment for the post-testicular sperm development in the epididymal lumen. The understanding of the biophysical properties of the epididymal epithelial cells is critical for revealing their functions in sperm and reproductive health, under both physiological and pathophysiological conditions. While their functional properties have yet to be fully elucidated, the epididymal epithelial cells can be studied using the patch-clamp technique, a tool for measuring the cellular events and the membrane properties of single cells. Here, we describe the methods of cell isolation and whole-cell patch-clamp recording to measure the electrical properties of primary dissociated epithelial cells from the rat cauda epididymides.

We present a protocol that combines cell isolation and whole-cell patch-clamp recording to measure the electrical properties of the primary dissociated epithelial cells from the rat cauda epididymides. This protocol allows for investigation of the functional properties of primary epididymal epithelial cells to further elucidate the physiological role of the epididymis.

The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types ...

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines retain the ability to either self-renew or differentiate under specific conditions. Due to these properties, mESC are a useful tool in regenerative medicine, disease modeling, and tissue engineering studies. This article describes a simple protocol to obtain mESC lines with high derivation efficiencies (60-80%) by culturing blastocysts from permissive mouse strains on feeder cells in defined medium supplemented with leukemia inhibitory factor. The protocol can also be applied to efficiently derive mESC lines from non-permissive mouse strains, by the simple addition of a cocktail of two small-molecule inhibitors to the derivation medium (2i medium). Detailed procedures on the preparation and culture of feeder cells, collection and culture of mouse embryos, and derivation and culture of mESC lines are provided. This protocol does not require specialized equipment and can be carried out in any laboratory with basic mammalian cell culture expertise.

This article describes a protocol to efficiently derive and culture pluripotent stem cell lines from mouse embryos at the blastocyst stage.

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines ...

AFM and Microrheology in the Zebrafish Embryo Yolk Cell

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. Various propositions claim to have been important contributions to the understanding of the mechanical properties of cells and tissues in their spatiotemporal organization in different developmental and morphogenetic processes. However, due to the lack of reliable and accessible tools to measure material properties and tensional parameters in vivo, validating these hypotheses has been difficult. Here we present methods employing atomic force microscopy (AFM) and particle tracking with the aim of quantifying the mechanical properties of the intact zebrafish embryo yolk cell during epiboly. Epiboly is an early conserved developmental process whose study is facilitated by the transparency of the embryo. These methods are simple to implement, reliable, and widely applicable since they overcome intrusive interventions that could affect tissue mechanics. A simple strategy was applied for the mounting of specimens, AFM recording, and nanoparticle injections and tracking. This approach makes these methods easily adaptable to other developmental times or organisms.

The lack of tools to measure material properties and tensional parameters in vivo prevents validating their roles during development. We employed atomic force microscopy (AFM) and nanoparticle-tracking to quantify mechanical features on the intact zebrafish embryo yolk cell during epiboly. These methods are reliable and widely applicable avoiding intrusive interventions.

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. ...

Application of Aorta-gonad-mesonephros Explant Culture System in Developmental Hematopoiesis

The limitation of using mouse embryos for hematopoiesis studies is the added inconvenience in operations, which is largely due to the intrauterine development of the embryo. Although genetic data from knockout (KO) mice are convincing, it is not realistic to generate KO mice for all genes as needed. In addition, performing in vivo rescue experiments to consolidate the data obtained from KO mice is not convenient. To overcome these limitations, the Aorta-Gonad-Mesonephros (AGM) explant culture was developed as an appropriate system to study hematopoietic stem cell (HSC) development. Especially for rescue experiments, it can be used to recover the impaired hematopoiesis in KO mice. By adding the appropriate chemicals into the medium, the impaired signaling can be reactivated or up-regulated pathways can be inhibited. With the use of this method, many experiments can be performed to identify the critical regulators of HSC development, including HSC related gene expression at mRNA and protein levels, colony formation ability, and reconstitution capacity. This series of experiments would be helpful in defining the underlying mechanisms essential for HSC development in mammals.

This protocol describes using cultured Aorta-Gonad-Mesonephros for expression analyses, colony-forming units in the culture and spleen, and long-term reconstitution to determine the effect of regulatory factors and signaling pathways on hematopoietic stem cell development. This has been demonstrated as an effective system for studying hematopoietic stem cell biology and function.

The limitation of using mouse embryos for hematopoiesis studies is the added inconvenience in operations, which is largely due to the intrauterine ...

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of the heart after ischemic injury. When activated, epicardial cells undergo a process known as epithelial to mesenchymal transition (EMT) to provide cells to the regenerating myocardium. Furthermore, the epicardium contributes via secretion of essential paracrine factors. To fully appreciate the regenerative potential of the epicardium, a human cell model is required. Here we outline a novel cell culture model to derive primary epicardial derived cells (EPDCs) from human adult and fetal cardiac tissue. To isolate EPDCs, the epicardium is dissected from the outside of the heart specimen and processed into a single cell suspension. Next, EPDCs are plated and cultured in EPDC medium containing the ALK 5-kinase inhibitor SB431542 to maintain their epithelial phenotype. EMT is induced by stimulation with TGF&#946;. This method enables, for the first time, the study of the process of human epicardial EMT in a controlled setting, and facilitates gaining more insight in the secretome of EPDCs that may aid heart regeneration. Furthermore, this uniform approach allows for direct comparison of human adult and fetal epicardial behavior.

The epicardium plays a crucial role in the development and repair of the heart by providing cells and growth factors to the myocardial wall. Here, we describe a method to culture human primary epicardial cells that enables the study and comparison of their developmental and adult characteristics.

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...