The authors thank UT AgResearch and the Department of Animal Science for supporting and optimizing this protocol. This work was funded by USDA grant.

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+crude+oil/dispersant+mixture+and+dispersant+components+on+PPAR+&#947;+activity+in+vitro+and+in+vivo:+identification+of+dioctyl+sodium+sulfosuccinate+(DOSS;+CAS#+577-11-7)+as+a+probable+obesogen.">Effects of crude oil/dispersant mixture and dispersant components on PPAR &#947; activity in vitro and in vivo: identification of dioctyl sodium sulfosuccinate (DOSS; CAS# 577-11-7) as a probable obesogen.</a> Environmental Health Perspectives. 124, 112-119 (2016).</li><li>Hausman, D. B., Park, H. J., Hausman, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Adipose Tissue Protocols. , 201-219 (2008).</li><li>Lee, M. J., Wu, Y., Fried, S. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Fungizone+in+control+of+fungi+and+yeasts+in+tissue+culture.">Use of Fungizone in control of fungi and yeasts in tissue culture.</a> Proceedings of the Society for Experimental Biology and Medicine. 106 (4), 880-883 (1961).</li><li>Kuhlmann, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prophylactic+use+of+antibiotics+in+cell+culture.">The prophylactic use of antibiotics in cell culture.</a> Cytotechnology. 19 (2), 95-105 (1995).</li><li>Yang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Black+swimming+dots+in+cell+culture:+the+identity,+detection+method+and+judging+criteria.">Black swimming dots in cell culture: the identity, detection method and judging criteria.</a> bioRxiv. , 366906 (2018).</li><li>Freshney, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culture of animal cells: a manual of basic technique and specialized applications. , 163-186 (2015).</li></ol>

The authors have nothing to disclose.

Although several well-described protocols have reported the isolation of preadipocytes14,15,16,17, isolation for embryonic preadipocytes has been optimized, which can be used for functional analyses of early life fat growth and development in broiler chicks. This protocol yields high viability embryonic adipocyte progenitors with high differentiation potential. Moreover, the presented procedure...

Obesity is a global health threat to both adults and children. Children who are overweight or obese are approximately five times more likely to be obese as adults, placing them at significantly increased risk for cardiovascular disease, diabetes, and many other comorbidities. About 13.4% of US children aged 2-5 have obesity1, illustrating that the tendency to accumulate excess body fat can be set in motion very early in life. For very different reasons, the accumulation of excess adipose tissue is a concern for broiler (meat-type) chickens. Modern broilers are incredibly efficient but still accumulate more lipid than is physiologically necessary2,3. This tendency begins soon after hatch and effectively wastes feed, the most expensive production component, by allocating it away from muscle growth. Therefore, for both children and broiler chickens, albeit for very different reasons, there is a need to understand factors that influence adipose tissue development and identify ways to limit adipose expansion early in life.
Adipocytes form from preadipocytes, adipose tissue-derived stem cells that undergo differentiation to develop mature, lipid-storing fat cells. Accordingly, preadipocytes in vitro are a valuable experimental model for obesity studies. These cells, isolated from the stromal vascular fraction of adipose depots, can provide a fundamental understanding of molecular pathways controlling adipocyte differentiation and metabolism4,5. Chick embryos are a favorable experimental model in developmental studies because culturing eggs on the desired schedule makes experimental manipulation easier, as it enables obtaining embryos without the mother's sacrifice to observe a series of developmental stages of embryos. Moreover, complicated surgical procedures and lengthy periods of time are not required to obtain embryos relative to larger animal models. Therefore, the chick embryo presents an opportunity to obtain preadipocytes from the earliest stages of adipose tissue development. Subcutaneous adipose tissue becomes visible in the chick around embryonic day 12 (E12) as a clearly defined depot located around the thigh. This depot is enriched in highly proliferative preadipocytes that actively undergo differentiation under developmental cues to form mature adipocytes6,7. The process of adipogenic differentiation is comparable between chickens and humans. Therefore, preadipocytes isolated from chick embryos can be used as a dual-purpose model for studies relevant to humans and poultry. However, the yield of preadipocytes declines with aging as cells grows into mature adipocytes5.
The present protocol optimizes the isolation of preadipocytes from adipose tissue during the stage (E16-E18) at which adipogenic differentiation and adipocyte hypertrophy are at their peak in broiler chick embryos8. This procedure can assess the effects of factors to which the developing embryo is exposed in ovo, such as the hen diet, on adipocyte development and adipogenic potential ex vivo. It can also test the impact of various manipulations (e.g., hypoxia, nutrient additions, pharmacological agonists, and antagonists) on adipogenesis or the various 'omes (e.g., transcriptome, metabolome, methylome) of adipocyte progenitors. As a representation of the earliest stage of adipose formation, cells obtained using this protocol are valuable models for studies relevant to poultry and humans.

<table><tbody><tr><td>1 mL Pipette</td><td>Eppendorf</td><td>Z683825</td><td>Single Channel Pipette, 100 - 1000 &amp;micro;L</td></tr><tr><td>1 mL Pipette Tip</td><td>Fisher Scientific</td><td>02-707-402</td><td></td></tr><tr><td>100% Isopropanol</td><td>Fisher Scientific</td><td>A426P4</td><td></td></tr><tr><td>1x PBS</td><td>Gibco</td><td>10010023</td><td></td></tr><tr><td>25 mL Flask</td><td>Pyrex</td><td>4980-25</td><td></td></tr><tr><td>37% Formaldehyde</td><td>Fisher Scientific</td><td>F75P-1GAL</td><td></td></tr><tr><td>6-Well Plate</td><td>Falcon</td><td>353046</td><td>Tissue Culture-treated</td></tr><tr><td>96-Well Assay Plate</td><td>Costar</td><td>3632</td><td></td></tr><tr><td>96-Well Plate, Black Bottom</td><td>Costar</td><td>3603</td><td>Tissue Culture-treated</td></tr><tr><td>AdipoRed</td><td>Lonza</td><td>PT-7009</td><td></td></tr><tr><td>Amphotericin B</td><td>Gibco</td><td>15290026</td><td></td></tr><tr><td>Bench Top Wiper (Kimtechwiper)</td><td>Kimberly-Clark</td><td>34155</td><td></td></tr><tr><td>Betadine</td><td>Up &amp;amp; Up</td><td>NDC 1167300334</td><td>20% Working Solution</td></tr><tr><td>Cell Counter</td><td>Corning</td><td>6749</td><td></td></tr><tr><td>Cell Strainer, 40 &amp;micro;m</td><td>SPL</td><td>93040</td><td></td></tr><tr><td>Centrifugaton</td><td>Eppendorf</td><td>5702</td><td></td></tr><tr><td>Chicken Serum</td><td>Gibco</td><td>16110082</td><td></td></tr><tr><td>Conical Centrifuge Tubes, 15 mL</td><td>VWR</td><td>10025-690</td><td></td></tr><tr><td>Conical Centrifuge Tubes, 50 mL</td><td>Falcon</td><td>352098</td><td></td></tr><tr><td>Cryovial</td><td>Nunc</td><td>343958</td><td></td></tr><tr><td>Curved Forceps, 100 mm</td><td>Roboz Surgical</td><td>RS-5137</td><td></td></tr><tr><td>Curved Surgical Scissors, 115 mm</td><td>Roboz Surgical</td><td>RS-6839</td><td></td></tr><tr><td>Distilled Water</td><td>Millipore</td><td>SYNSV0000</td><td>Despensed as needed</td></tr><tr><td>DMEM/F12</td><td>HyClone</td><td>SH30023.01</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>Ethanol</td><td>Decon Labs</td><td>2701</td><td>70% Working Solution</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco</td><td>10437028</td><td></td></tr><tr><td>Fluorescent Microscope</td><td>EVOS</td><td>M7000</td><td></td></tr><tr><td>Fluorescent Plate Reader</td><td>Biotek</td><td>Synergy H1</td><td></td></tr><tr><td>Foil</td><td>Reynolds</td><td></td><td>Reynolds Wrap Heavy Duty Aluminum Foil, 125 SQ. FT.</td></tr><tr><td>Freezing Container</td><td>Thermo Scientific</td><td>5100-0001</td><td></td></tr><tr><td>Gelatin</td><td>Millipore</td><td>4055</td><td>2% Working Solution</td></tr><tr><td>Hematocytometer (Counting Chamber)</td><td>Corning</td><td>480200</td><td>0.1 mm deep</td></tr><tr><td>Incubator</td><td>Fisher Scientific</td><td>6845</td><td></td></tr><tr><td>Instrument Sterilizer</td><td>VWR</td><td>B1205</td><td></td></tr><tr><td>Linoleic Acid-Oleic Acid-Albumin</td><td>Sigma</td><td>L9655</td><td>1x Working Solution</td></tr><tr><td>Microscope</td><td>Evos</td><td>AMEX1000</td><td></td></tr><tr><td>Multi-Channel Pipette</td><td>Thermo Scientific</td><td>4661070</td><td>12-Channel Pipetters, 30 - 300 &amp;micro;L</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>S-7907</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>S-3139</td><td></td></tr><tr><td>NucBlue</td><td>Invitrogen</td><td>R37605</td><td></td></tr><tr><td>Oil Red O</td><td>Sigma</td><td>O-0625</td><td></td></tr><tr><td>Orbital Shaker</td><td>IKA</td><td>KS130BS1</td><td></td></tr><tr><td>Paper Towel</td><td>Tork</td><td>RK8002</td><td></td></tr><tr><td>Parafilm</td><td>Parafilm M</td><td>PM996</td><td></td></tr><tr><td>Penicillin/Steptomycin (P/S)</td><td>Gibco</td><td>15140122</td><td>1x Working Solution</td></tr><tr><td>Petri dishes, 100 mm</td><td>Falcon</td><td>351029</td><td></td></tr><tr><td>Petri dishes, 60 mm</td><td>Falcon</td><td>351007</td><td></td></tr><tr><td>Plate Shaker</td><td>VWR</td><td>200</td><td></td></tr><tr><td>RBC Lysis Buffer</td><td>Roche</td><td>11814389001</td><td></td></tr><tr><td>Reagent Reservior</td><td>VWR</td><td>89094-680</td><td></td></tr><tr><td>Small Beaker, 100 mL</td><td>Pyrex</td><td>1000-100</td><td></td></tr><tr><td>Spectrophotometer Plate Reader</td><td>Biotek</td><td>Synergy H1</td><td></td></tr><tr><td>Sterile Gauze</td><td>McKesson</td><td>762703</td><td></td></tr><tr><td>Straight Forceps, 120 mm</td><td>Roboz Surgical</td><td>RS-4960</td><td></td></tr><tr><td>Straight Scissors, 140 mm</td><td>Roboz Surgical</td><td>RS-6762</td><td></td></tr><tr><td>T-25 Flask</td><td>Corning</td><td>430639</td><td>Tissue Culture-treated</td></tr><tr><td>Tissue Culture Incubator</td><td>Thermo Scientific</td><td>50144906</td><td></td></tr><tr><td>Tissue Strainer, 250 &amp;micro;m</td><td>Pierce</td><td>87791</td><td></td></tr><tr><td>Trypan Blue Stain</td><td>Gibco</td><td>15250061</td><td></td></tr><tr><td>Trypsin</td><td>Gibco</td><td>15400054</td><td>0.1% Working Solution</td></tr><tr><td>Tweezers, 110 mm</td><td>Roboz Surgical</td><td>RS-5035</td><td></td></tr><tr><td>Type 1 Collagenase</td><td>Gibco</td><td>17100017</td><td></td></tr><tr><td>Water Bath</td><td>Fisher Scientific</td><td>15-462-10</td><td></td></tr><tr><td>Whatman Grade 1 Filter Paper</td><td>Whatman</td><td>1001-110</td><td></td></tr></tbody></table>

isolation of preadipocytes from broiler chick embryos

Primary preadipocytes are a valuable experimental system for understanding the molecular pathways that control adipocyte differentiation and metabolism. Chicken embryos provide the opportunity to isolate preadipocytes from the earliest stage of adipose development. This primary cell can be used to identify factors influencing preadipocyte proliferation and adipogenic differentiation, making them a valuable model for studies related to childhood obesity and control of excess fat deposition in poultry. The rapid growth of postnatal adipose tissue effectively wastes feed by allocating it away from muscle growth in broiler chickens. Therefore, methods to understand the earliest stages of adipose tissue development may provide clues to regulate this tendency and identify ways to limit adipose expansion early in life. The present study was designed to develop an efficient method for isolation, primary culture, and adipogenic differentiation of preadipocytes isolated from developing adipose tissue of commercial broiler (meat-type) chick embryos. The procedure has been optimized to yield cells with high viability (~98%) and increased capacity to differentiate into mature adipocytes. This simple method of embryonic preadipocyte isolation, culture, and differentiation supports functional analyses of fat growth and development in early life.

Primary preadipocytes are morphologically similar to fibroblasts, with irregular, star-like shapes and a central nucleus (Figure 2A-C). The cells readily adhere to tissue culture plastic and begin to proliferate soon after attachment. They rapidly differentiate and accumulate lipid droplets (Figure 3D) when provided with fatty acids in the media. The viability (98%, based on dye exclusion) reported in the isolations represented ...

Watch this Scientific Journal Video about Isolation of Preadipocytes from Broiler Chick Embryos at JoVE.com

Isolation of Preadipocytes from Broiler Chick Embryos

The present protocol describes a simple method for isolating preadipocytes from adipose tissue in broiler embryos. This method enables isolation with high yield, primary culture, and adipogenic differentiation of preadipocytes. Oil Red O staining and lipid/DNA stain measured the adipogenic ability of isolated cells induced with differentiation media.

Primary preadipocytes are a valuable experimental system for understanding the molecular pathways that control adipocyte differentiation and metabolism. ...

isolation-of-preadipocytes-from-broiler-chick-embryos

Research

JoVE Journal

Developmental Biology

2.2K Views.  University of Tennessee. The present protocol describes a simple method for isolating preadipocytes from adipose tissue in broiler embryos. This method enables isolation with high yield, primary culture, and adipogenic differentiation of preadipocytes. Oil Red O staining and lipid/DNA stain measured the adipogenic ability of isolated cells induced with differentiation media.

Video: Isolation of Preadipocytes from Broiler Chick Embryos

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin-modifying enzymes at a given locus or on a genome-wide scale. The combination of ChIP assays with next-generation sequencing (i.e., ChIP-Seq) is a powerful approach to globally uncover gene regulatory networks and to improve the functional annotation of genomes, especially of non-coding regulatory sequences. ChIP protocols normally require large amounts of cellular material, thus precluding the applicability of this method to investigating rare cell types or small tissue biopsies. In order to make the ChIP assay compatible with the amount of biological material that can typically be obtained in vivo during early vertebrate embryogenesis, we describe here a simplified ChIP protocol in which the number of steps required to complete the assay were reduced to minimize sample loss. This ChIP protocol has been successfully used to investigate different histone modifications in various embryonic chicken and adult mouse tissues using low to medium cell numbers (5 x 104 - 5 x 105 cells). Importantly, this protocol is compatible with ChIP-seq technology using standard library preparation methods, thus providing global epigenomic maps in highly relevant embryonic tissues.

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

Here, we describe a chromatin immunoprecipitation (ChIP) and ChIP-seq library preparation protocol to generate global epigenomic profiles from low-abundance chicken embryonic samples.

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, ...

Genetics

Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be involved in differentially regulating cancer metastatic cascade genes and gene products dependent on the cancer type, tissue, and individual patient characteristics. These represent potentially important targets for genetic therapeutics and personalized medicine approaches. The development of rapid screening platforms is essential for the identification of these genetic targets.
The chick chorioallantoic membrane (CAM) is a highly vascularized, collagen rich membrane located under the eggshell that allows for gas exchange in the developing embryo. Due to the location and vascularization of the CAM, we developed it as an intravital human cancer metastasis model that allows for robust human cancer cell xenografting and real-time imaging of cancer cell interactions with the collagen rich matrix and vasculature. 
Using this model, a quantitative screening platform was designed for the identification of novel drivers or suppressors of cancer metastasis. We transduced a pool of head and neck HEp3 cancer cells with a complete human genome shRNA gene library, then injected the cells, at low density, into the CAM vasculature. The cells proliferated and formed single-tumor cell colonies. Individual colonies that were unable to invade into the CAM tissue were visible as a compact colony phenotype and excised for identification of the transduced shRNA present in the cells. Images of individual colonies were evaluated for their invasiveness. Multiple rounds of selections were performed to decreases the rate of false positives. Individual, isolated cancer cell clones or newly engineered clones that express genes of interest were subjected to primary tumor formation assay or cancer cell vasculature co-option analysis. In summary we present a rapid screening platform that allows for anti-metastatic target identification and intravital analysis of a dynamic and complex cascade of events.

This is an effective method to screen for suppressors or drivers of cancer metastasis. Cells, transduced with an expression library, are injected into the chicken chorioallantoic membrane vasculature to form metastatic colonies. Colonies having decreased or increased invasiveness are excised, expanded, reinjected to confirm their phenotype, and finally, analyzed using high throughput sequencing.

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be ...

Cancer Research

Isolation of Embryonic Tissues and Formation of Quail-Chicken Chimeric Organs Using The Thymus Example

The capacity to isolate embryonic tissues was an essential step for establishing the quail-chicken chimera system, which in turn has provided undisputed contributions to unveiling key processes in developmental biology.
Herein is described an optimized method to isolate embryonic tissues from quail and chickens by microsurgery and enzymatic digestion while preserving its biological properties. After isolation, tissues from both species are associated in an in vitro organotypic assay for 48 h. Quail and chicken tissues can be discriminated by distinct nuclear features and molecular markers allowing the study of the cellular cross-talk between heterospecific association of tissues. This approach is, therefore, a useful tool for studying complex tissue interactions in developmental processes with highly dynamic spatial modifications, such as those occurring during pharyngeal morphogenesis and the formation of the foregut endoderm-derived organs. This experimental approach was first developed to study the epithelial-mesenchymal interactions during early-stages of thymus formation. In this, the endoderm-derived prospective thymic rudiment and mesoderm-derived mesenchyme, were isolated from quail and chicken embryos, respectively.
The capacity of the associated tissues to generate organs can be further tested by grafting them onto the chorioallantoic membrane (CAM) of a chicken embryo. The CAM provides nutrients and allows gas exchanges to the explanted tissues. After 10 days of in ovo development, the chimeric organs can be analyzed in the harvested explants by conventional morphological methods. This procedure also allows studying tissue-specific contributions during organ formation, from its initial development (in vitro development) to the final stages of organogenesis (in ovo development).
Finally, the improved isolation method also provides three-dimensionally (3D) preserved embryonic tissues, that can also be used for high-resolution topographical analysis of tissue-specific gene-expression patterns.

This article provides a method to isolate pure embryonic tissues from quail and chicken embryos that can be combined to form ex vivo chimeric organs.

The capacity to isolate embryonic tissues was an essential step for establishing the quail-chicken chimera system, which in turn has provided undisputed ...

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. Studying neurons in vivo can be challenging due to their complexity, their varied and dynamic environments, and technical limitations. For these reasons, studying neurons in vitro can prove beneficial to unravel the complex mysteries of neurons. The well-defined nature of cell culture models provides detailed control over environmental conditions and variables. Here we describe how to isolate, dissociate, and culture primary neurons from chick embryos. This technique is rapid, inexpensive, and generates robustly growing sensory neurons. The procedure consistently produces cultures that are highly enriched for neurons and has very few non-neuronal cells (less than 5%). Primary neurons do not adhere well to untreated glass or tissue culture plastic, therefore detailed procedures to create two distinct, well-defined laminin-containing substrata for neuronal plating are described. Cultured neurons are highly amenable to multiple cellular and molecular techniques, including co-immunoprecipitation, live cell imagining, RNAi, and immunocytochemistry. Procedures for double immunocytochemistry on these cultured neurons have been optimized and described here.

Cell culture models provide detailed control over environmental conditions and thus provide a powerful platform to elucidate numerous aspects of neuronal cell biology. We describe a rapid, inexpensive, and reliable method to isolate, dissociate, and culture sensory neurons from chick embryos. Details of substrata preparation and immunocytochemistry are also provided.

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. ...

Neuroscience

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

Biology

Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident adipose progenitor cells (APCs) play a key role in adipose tissue homeostasis and can contribute to the tissue pathology. The growing use of single cell analysis technologies &#8211; including single-cell RNA-sequencing and single-cell proteomics &#8211; is transforming the stem/progenitor cell field by permitting unprecedented resolution of individual cell expression changes within the context of population- or tissue-wide changes. In this article, we provide detailed protocols to dissect mouse epididymal adipose tissue, isolate single adipose tissue-derived cells, and perform fluorescence activated cell sorting (FACS) to enrich for viable Sca1+/CD31-/CD45-/Ter119- APCs. These protocols will allow investigators to prepare high quality APCs suitable for downstream analyses such as single cell RNA sequencing.

We present a simple method to isolate highly viable adipose progenitor cells from mouse epididymal fat pads using fluorescence activated cell sorting.

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident ...

Isolation and Characterization of Human Adipocyte-Derived Extracellular Vesicles using Filtration and Ultracentrifugation

Extracellular vesicles (EVs) are lipid enclosed envelopes that carry biologically active material such as proteins, RNA, metabolites and lipids. EVs can modulate the cellular status of other cells locally in tissue microenvironments or through liberation into peripheral blood. Adipocyte-derived EVs are elevated in the peripheral blood and show alterations in their cargo (RNA and protein) during metabolic disturbances, including obesity and diabetes. Adipocyte-derived EVs can regulate the cellular status of neighboring vascular cells, such as endothelial cells and adipose tissue resident macrophages to promote adipose tissue inflammation. Investigating alterations in adipocyte-derived EVs in vivo is complex because EVs derived from peripheral blood are highly heterogenous and contain EVs from other sources, namely platelets, endothelial cells, erythrocytes and muscle. Therefore, the culture of human adipocytes provides a model system for the study of adipocyte derived EVs. Here, we provide a detailed protocol for the extraction of total small EVs from cell culture media of human gluteal and abdominal adipocytes using filtration and ultracentrifugation. We further demonstrate the use of Nanoparticle Tracking Analysis (NTA) for quantification of EV size and concentration and show the presence of EV-protein tumor susceptibility gene 101 (TSG101) in the gluteal and abdominal adipocyte derived-EVs. Isolated EVs from this protocol can be used for downstream analysis, including transmission electron microscopy, proteomics, metabolomics, small RNA-sequencing,&#160;microarrays and can be&#160;utilized in functional in vitro/in vivo studies.

We describe the isolation of human adipocyte-derived extracellular vesicles (EVs) from gluteal and abdominal adipose tissue using filtration and ultracentrifugation. We characterize the isolated adipocyte-derived EVs by determining their size and concentration by Nanoparticle Tracking Analysis and by western blotting for the presence of EV-protein tumor susceptibility gene 101 (TSG101).

Extracellular vesicles (EVs) are lipid enclosed envelopes that carry biologically active material such as proteins, RNA, metabolites and lipids. EVs can ...

Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white preadipocyte cell lines. These cultured cell lines, however, have limitations. They do not fully capture the diverse functional spectrum of the heterogenous adipocyte populations that are now known to exist within white adipose depots. To provide a more physiologically relevant model to study the complexity of white adipose tissue, a protocol has been developed and optimized to enable simultaneous isolation of primary white and brown adipocyte progenitors from newborn mice, their rapid expansion in culture, and their differentiation in vitro into mature, fully functional adipocytes. The primary advantage of isolating primary cells from newborn, rather than adult mice, is that the adipose depots are actively developing and are, therefore, a rich source of proliferating preadipocytes. Primary preadipocytes isolated using this protocol differentiate rapidly upon reaching confluence and become fully mature in 4-5 days, a temporal window that accurately reflects the appearance of developed fat pads in newborn mice. Primary cultures prepared using this strategy can be expanded and studied with high reproducibility, making them suitable for genetic and phenotypic screens and enabling the study of the cell-autonomous adipocyte phenotypes of genetic mouse models. This protocol offers a simple, rapid, and inexpensive approach to study the complexity of adipose tissue in vitro.

This report describes a protocol for the simultaneous isolation of primary brown and white preadipocytes from newborn mice. Isolated cells can be grown in culture and induced to differentiate into fully mature white and brown adipocytes. The method enables genetic, molecular, and functional characterization of primary fat cells in culture.

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white ...

Semi-Automated Isolation of Murine White Adipose Tissue

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their mechanisms. Immortalized white preadipocyte cell lines are publicly available and widely used. However, the emergence of beige adipocytes in white adipose tissue in response to external cues is difficult to recapitulate to the full extent using publicly available white adipocyte cell lines. Isolation of the stromal vascular fraction (SVF) from murine adipose tissue is commonly executed to obtain primary preadipocytes and perform adipocyte differentiation. However, mincing and collagenase digestion of adipose tissue by hand can result in experimental variation and is prone to contamination. Here, we present a modified semi-automated protocol that utilizes a tissue dissociator for collagenase digestion to achieve easier isolation of the SVF, with the aim of reducing experimental variation, reducing contamination, and increasing reproducibility. The obtained preadipocytes and differentiated adipocytes can be used for functional and mechanistic analyses.

Author Spotlight: Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator  

This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility.

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their ...

Separation of Hen Ovarian Follicle Layers for Reproductive Research Applications

Separation of Avian Preovulatory Follicle Granulosa and Theca Cell Layers for Downstream Applications

Layer hens (egg-laying chickens) and broiler breeders (breeding stock for meat-producing chickens) are crucial to the world's food supply as a reliable source of protein. They are also an emerging animal model for the study of human reproductive disease. As the field of poultry research develops, the health and function of the layer hen and broiler breeder ovary will be an important point of study for both agricultural and biomedical researchers. One of the challenges presented by this emerging interest is the need for replicable techniques that all researchers can employ in ovarian specimen collection. In particular, a detailed visual process must be established to define the proper separation of the specialized granulosa and theca cell layers from hen follicles to achieve agreement and consistency among researchers. 
This study describes the extraction of preovulatory follicles and ovary tissue in white leghorn hens of prime reproductive age. The separation of these follicles is performed under cold, liquid conditions to congeal the yolk for easier manipulation and to prevent the follicle's own weight from tearing apart cell layers during the separation process. Once the separation is complete, the desired cell layers can be further digested for tissue culture approaches or can be cryopreserved for genomic and proteomic analyses.

Here, we describe a protocol for separating yolk, granulosa cells, and theca cells in avian preovulatory follicles. This precision handling enables critical investigations into the role of these layers in reproductive function, aiding the understanding of follicular development, hormonal regulation, and disease research for enhanced agricultural yield and biomedical insights.

Layer hens (egg-laying chickens) and broiler breeders (breeding stock for meat-producing chickens) are crucial to the world's food supply as a reliable ...