Current protocols最新文献

{"title":"Using Vesicular Stomatitis Virus as a Platform for Directed Protease Evolution","authors":"Francesco Costacurta,&nbsp;Stefanie Rauch,&nbsp;Dorothee von Laer,&nbsp;Emmanuel Heilmann","doi":"10.1002/cpz1.70074","DOIUrl":"10.1002/cpz1.70074","url":null,"abstract":"<p>Antiviral drugs are essential medications to save the lives of infected people. However, they are under constant threat to become ineffective as viruses evolve quickly. Studying the development of resistance is therefore paramount to understand the impact of mutations on pharmacological treatment and to make informed decisions. Yet, such studies are open to scrutiny, as they are considered gain-of-function research, which is especially problematic with viruses of pandemic potential. In this article, we present a protocol that allows for the selection of antiviral resistance mutations safely, without using the actual virus (e.g., SARS-CoV-2, MERS-CoV). Instead, we use vesicular stomatitis virus (VSV) that serves as a surrogate virus; like other RNA viruses, it is prone to mutations due to its polymerase lacking proofreading. By replacing parts of the VSV genome with transgenes from other viruses, VSV becomes dependent on their function. Thus, we can mount a selection pressure with antivirals targeting the transgenes to subsequently sequence selected resistance mutations. This article provides a protocol for this process as well as a sequencing pipeline that we used to collect mutations. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Using VSV as a platform for directed protease evolution</p><p><b>Alternate Protocol</b>: Dose response assay with TCID₅₀ readout</p><p><b>Support Protocol 1</b>: A pipeline for high-throughput VSV sequencing</p><p><b>Support Protocol 2</b>: Rescue of VSV</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 12","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-12-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11664493/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142879210","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Employment of a Newly Defined In Vitro Fertilization Protocol to Determine the Cytoskeletal Machinery, DNA Damage, and Subsequent DNA Repair Resulting from Endocrine Disruption by Hexavalent Chromium in Rat Metaphase II Oocytes","authors":"Liga Wuri,&nbsp;Paul W. Zarutskie,&nbsp;Joe A. Arosh,&nbsp;Sakhila K. Banu","doi":"10.1002/cpz1.70060","DOIUrl":"10.1002/cpz1.70060","url":null,"abstract":"<p>These protocols describe a detailed method to determine the DNA damage and F-actin and microtubule defects of metaphase II oocytes caused by hexavalent chromium, Cr(VI), an endocrine disrupting chemical (EDC). The protocol provides systematic steps to determine protein expression encoded by pluripotency proteins such as Oct4, Nanog, and Cdx2 during early embryonic development. Occupational or environmental exposure to EDCs has significantly increased infertility in both men and women. The urinary concentration of the EDC bisphenol A in patients undergoing <i>in vitro</i> fertilization (IVF) is directly related to decreased implantation rates and the number of metaphase II oocytes recovered. This protocol outlines crucial steps in assessing the structure of F-actin and microtubules, DNA damage, and repair mechanisms in metaphase II oocytes as well as pluripotency protein markers of early-stage embryos. IVF techniques to achieve fertility goals in both humans and animals are of paramount importance. The interplay between F-actin and microtubules is crucial for bipolar spindle assembly and correct partitioning of the nuclear genome in mammalian oocyte meiosis. EDCs induce DNA damage and impair DNA repair mechanisms, compromising oocyte quality. In human IVF, this results in failure to implant, early miscarriage, and live births with congenital disorders, thus decreasing success rates and increasing poor outcomes. The application of IVF protocols in rats to understand EDC-mediated defects in the cytoskeletal network of metaphase II oocytes is not well established. We present a newly defined rat IVF protocol and demonstrate outcomes using these protocols to determine the adverse effects of Cr(VI) on metaphase II oocytes. Basic Protocol 1 includes steps to superovulate rats, dissect ampullae, retrieve oocytes/eggs, perform immunofluorescence staining of cytoskeletal machinery (microtubules and F-actin), and assess expression of the DNA double-strand break marker γ-H2AX and the DNA repair protein RAD51 in control and Cr(VI)-exposed rats. Basic Protocol 2 describes methods for detecting the pluripotency proteins Oct4, Nanog, and Cdx2 during early embryonic development in control rats. © 2024 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: <i>In vivo</i> EDC treatment of rats and immunostaining of treated oocytes</p><p><b>Basic Protocol 2</b>: <i>In vitro</i> fertilization and immunostaining of early-stage embryos</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 12","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-12-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142879207","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Isolation, Purification, and Comprehensive Flow Cytometry Assessment of Lung Stromal Cells","authors":"Sophia Rottmann,&nbsp;Veronika Lukacs-Kornek","doi":"10.1002/cpz1.70078","DOIUrl":"10.1002/cpz1.70078","url":null,"abstract":"<p>Stromal cells are non-hematopoietic cells that consist of endothelial cells and various mesenchymal cell populations. The composition of the stromal cell compartment is diverse in different organs. Numerous recent studies demonstrated that the lung environment contains heterogeneous mesenchymal stromal cell populations with distinctive genomic signatures and location preferences. Besides their role in supporting organ structure and remodeling tissue, mesenchymal stromal cells fulfill critical immune functions. These stromal cells show alterations during lung fibrosis and infectious disorders like COVID-19 or flu infection.</p><p>To date, their identification and isolation were challenging, and most information about their heterogeneity was derived from scRNAseq data. In this protocol, we describe an isolation, comprehensive flow cytometry assessment, and purification strategy for murine lung stromal cells. The described method is optimized for minimizing cell death while keeping a high level of cell purity. This protocol can be also used for ex-vivo analysis of these cells in downstream functional assays. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Isolation of stromal cells from murine lung tissue</p><p><b>Basic Protocol 2</b>: Flow cytometry assessment of lung stromal populations</p><p><b>Basic Protocol 3</b>: Purification of lung fibroblastic stromal cells</p><p><b>Alternate Protocol</b>: Positive selection of fibroblastic stromal cells</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 12","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70078","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142857314","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of a Microphysiological Cartilage-on-Chip Platform for Dynamic Biomechanical Stimulation of Three-Dimensional Encapsulated Chondrocytes in Agarose Hydrogels","authors":"Valtteri Peitso,&nbsp;Zahra Sarmadian,&nbsp;João Henriques,&nbsp;Elsa Lauwers,&nbsp;Carlo Alberto Paggi,&nbsp;Ali Mobasheri","doi":"10.1002/cpz1.70079","DOIUrl":"10.1002/cpz1.70079","url":null,"abstract":"<p>Osteoarthritis (OA) is one of the most prevalent joint diseases globally, characterized by the progressive breakdown of articular cartilage, resulting in chronic pain, stiffness, and loss of joint function. Despite its significant socioeconomic impact, therapeutic options remain limited, largely due to an incomplete understanding of the molecular mechanisms driving cartilage degradation and OA pathogenesis. Recent advances in <i>in vitro</i> modeling have revolutionized joint tissue research, transitioning from simplistic two-dimensional cell cultures to sophisticated three-dimensional (3D) constructs that more accurately mimic the physiological microenvironment of native cartilage. Over the last decade, organ-on-chip technologies have emerged as transformative tools in tissue engineering, offering microphysiological platforms with precise control over biomechanical and biochemical stimuli. These platforms are providing novel insights into tissue responses and disease progression and are increasingly integrated into the early stages of drug screening and development. In this article, we present a detailed experimental protocol for constructing a cartilage-on-chip system capable of delivering controlled dynamic biomechanical stimulation to 3D-encapsulated chondrocytes in an agarose hydrogel matrix. Our protocol, optimized for both bovine and human chondrocytes, begins with Basic Protocol 1, detailing the preparation and injection of cell-laden hydrogels into the microdevice. Basic Protocol 2 describes the application of dynamic mechanical loading using a calibrated pressurized pump. Finally, Basic Protocols 3 and 4 focus on the retrieval of the hydrogel and RNA extraction for downstream molecular analyses. This platform represents a critical advancement for <i>in vitro</i> studies of cartilage biology, enabling more precise modeling of OA pathophysiology and evaluation of experimental therapeutics. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Cartilage-on-chip injection</p><p><b>Basic Protocol 2</b>: Cartilage-on-chip actuation</p><p><b>Basic Protocol 3</b>: Cartilage-on-chip agarose hydrogel removal</p><p><b>Basic Protocol 4</b>: Preparation of cartilage-on-chip for RNA extraction</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 12","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70079","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142857311","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Comprehensive Stereology Method for Quantitative Evaluation of Neuronal Injury, Neurodegeneration, and Neurogenesis in Brain Disorders","authors":"Doodipala Samba Reddy,&nbsp;Neo Zhu,&nbsp;Trisha Challa,&nbsp;Sai Gajjela,&nbsp;Hetvi Desai,&nbsp;Sreevidhya Ramakrishnan,&nbsp;Xin Wu","doi":"10.1002/cpz1.70053","DOIUrl":"10.1002/cpz1.70053","url":null,"abstract":"&lt;p&gt;Neuronal injury, neurodegeneration, and neuroanatomical changes are key pathological features of various neurological disorders, including epilepsy, stroke, traumatic brain injury, Parkinson's disease, autism, and Alzheimer's disease. Accurate quantification of neurons and interneurons in different brain regions is critical for understanding the progression of neurodegenerative disorders in animal models. Traditional scoring methods are often superficial, biased, and unreliable for evaluating neuropathology. Stereology, a quantitative tool that uses 3-dimensional visualization of cells, provides a robust protocol for evaluating neuronal injury and neurodegeneration. This article presents a comprehensive and optimized stereology protocol for unbiased quantification of neuronal injury, neurodegeneration, and neurogenesis in rat and mouse models. This protocol involves precise counting of injured neurons, surviving neurons, and interneurons through immunohistochemical processing of brain sections for NeuN(+) principal neurons, parvalbumin (PV+) interneurons, doublecortin (DCX+) newborn neurons, and Fluoro-Jade B (FJB+)-stained injured cells. Predefined hippocampal and amygdala regions were identified and analyzed using a Visiopharm stereology software-driven compound microscope. Cell density and absolute cell numbers were determined using the optical fractionation method. Our stereology protocol accurately estimated 1.5 million total NeuN(+) principal neurons and 0.05 million PV(+) interneurons in the rat hippocampus, as well as 1.2 million total principal neurons and 0.025 million interneurons in the mouse hippocampus. FJB(+) counting provided a quantitative index of damaged neurons, and the stereology of DCX(+) neurons demonstrated the extent of neurogenesis. Overall, this stereology protocol enables precise, accurate, and unbiased counting of total neurons in any brain region. This offers a reliable quantitative tool for studying neuronal injury and protection in various models of acute brain injury, neurotoxicity, and chronic neurological disorders. © 2024 Wiley Periodicals LLC.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Basic Protocol 1&lt;/b&gt;: Stereological quantification of principal neurons, interneurons, and immature neurons in the hippocampus in rat brain sections&lt;/p&gt;&lt;p&gt;&lt;b&gt;Basic Protocol 2&lt;/b&gt;: Stereological quantification of principal neurons, interneurons, and immature neurons in the hippocampus in mouse brain sections&lt;/p&gt;&lt;p&gt;&lt;b&gt;Basic Protocol 3&lt;/b&gt;: Stereological quantification of injured or necrotized cells stained with Fluoro-Jade B in the hippocampus and amygdala in rats&lt;/p&gt;&lt;p&gt;&lt;b&gt;Basic Protocol 4&lt;/b&gt;: Stereological quantification of injured or necrotized cells stained with Fluoro-Jade B in the hippocampus and amygdala regions in mice&lt;/p&gt;&lt;p&gt;&lt;b&gt;Basic Protocol 5&lt;/b&gt;: Brain fixation and histology processing&lt;/p&gt;&lt;p&gt;&lt;b&gt;Basic Protocol 6&lt;/b&gt;: Immunochemistry of principal neurons, interneurons, and newborn neurons&lt;/p&gt;&lt;p&gt;&lt;b&gt;Basic Protocol 7&lt;/b&gt;: Fluoro-Jade B staining of injured","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 12","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142857305","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Biotin-Based Northern Blotting (BiNoB): A Cost-Efficient Alternative for Detection of Small RNAs","authors":"Deeksha Madhry,&nbsp;Riya Roy,&nbsp;Bhupendra Verma","doi":"10.1002/cpz1.70065","DOIUrl":"10.1002/cpz1.70065","url":null,"abstract":"<p>Advances in sequencing technology have led to the discovery of diverse types of regulatory RNAs. Differential transcript levels regulate cellular processes and influence disease severity. Identifying these variations through reliable methods is crucial for understanding the regulatory roles and disease mechanisms of regulatory RNAs. Northern blotting, which is considered the gold standard for differential expression analysis, poses challenges due to various limitations associated with RNA quality and integrity, radioactivity exposure, and associated reagents and expenses. In this protocol, we employ a biotin-based northern blotting (BiNoB) approach that is both convenient and inexpensive, eliminating the need for specialized settings as required with radioactivity-based northern blotting. We comprehensively target various RNA types, making this technique a versatile tool for RNA detection. Additionally, we conduct a comparison between 3′-end labeled probes that were labeled in-house and 5′-end labeled probes that were obtained commercially. Remarkably, our results reveal relatively higher sensitivity with 3′-end labeled probes. Furthermore, we demonstrated that the use of an in-house buffer offered comparable sensitivity to a commercially available buffer, providing another cost-effective alternative. We also aimed to determine the minimum quantity of total RNA required to detect small non-coding RNAs such as tRNA fragments. Whereas previous studies reported the use of 5-10 µg total RNA for tRNA fragment detection, our findings revealed that as little as 1 µg total RNA is sufficient to detect small RNAs like tRNAs and their fragments. This concentration may vary depending on the expression levels of the specific RNAs being detected. © 2024 Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Biotin-based northern blotting</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 12","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142857308","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Quantifying Biomass and Visualizing Cell Coverage on Fibrous Scaffolds for Cultivated Meat Production","authors":"Xinxin Li,&nbsp;Khin Thu Lwin,&nbsp;Hirunika U. Kumarasinghe,&nbsp;Lilianne Iglesias-Ledon,&nbsp;Eesha Bethi,&nbsp;Yushu Wang,&nbsp;Colin Fennelly,&nbsp;Ryan Sylvia,&nbsp;Sonja Hatz,&nbsp;Timothy Olsen,&nbsp;Thomas Herget,&nbsp;Ying Chen,&nbsp;David Kaplan","doi":"10.1002/cpz1.70076","DOIUrl":"10.1002/cpz1.70076","url":null,"abstract":"<p>Cultivated meat represents a transformative solution to environmental and ethical concerns of traditional meat industries, replicating livestock meat's texture and sensory attributes in vitro with a focus on cost, safety, and nutritional quality. Central to this process are biomaterial scaffolds that support tissue development from isolated animal cells grown in or on these matrices. Understanding scaffold interactions with cells, including scaffold degradation and biomass production, is crucial for process design and for scaling-up goals. In this article, we outline comprehensive methods to quantify scaffold-cell interactions for such scenarios, focusing on biomaterial scaffold degradation and changes in cell biomass [measured by cell weight, extracellular matrix (ECM) deposition, and cell coverage] during cell culture. We introduce two methodologies for assessing cell coverage: fixation and staining for detailed imaging analysis, and non-invasive, real-time evaluation across scaffolds. Here we focus on fiber-based scaffolds, while the assessments can be extrapolated to 2-dimensional (2D; films), and in part to 3-dimensional (3D; sponge) systems. Utilizing the C2C12 mouse myoblast cell line as a gold standard, the protocols deliver precise, step-by-step instructions for preparing fiber scaffolds (using silk proteins here), seeding cells, and monitoring key parameters for cultivated meat production, providing a framework for advancing cellular agriculture techniques. © 2024 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Fabrication and preparation of silk fiber scaffolds for cell seeding</p><p><b>Support Protocol 1</b>: Cultivation of C2C12 cells and seeding onto fibrous scaffolds</p><p><b>Basic Protocol 2</b>: Quantification of decellularized yarn scaffold degradation during cell culture</p><p><b>Basic Protocol 3</b>: Quantification of biomass variation and ECM deposition on yarn scaffolds during C2C12 cell culture</p><p><b>Basic Protocol 4</b>: Visualization of cell-laden yarn scaffolds and determination of cell coverage ratio using confocal microscopy</p><p><b>Support Protocol 2</b>: Real-time imaging of cell-laden yarn scaffolds using Celigo system</p><p><b>Support Protocol 3</b>: Applying green CellTracker fluorescent probes to C2C12 cells seeded on yarn scaffolds</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 12","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142857324","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Correction: Functional Assays Evaluating Immunosuppression Mediated by Myeloid-Derived Suppressor Cells","authors":"Or Reuven,&nbsp;Ivan Mikula Jr.,&nbsp;Hadas Ashkenazi-Preiser,&nbsp;Nira Twaik,&nbsp;Kerem Ben-Meir,&nbsp;Yaron Meirow,&nbsp;Leonor Daniel,&nbsp;Guy Kariv,&nbsp;Mahdi Kurd,&nbsp;Michal Baniyash","doi":"10.1002/cpz1.70082","DOIUrl":"10.1002/cpz1.70082","url":null,"abstract":"<p><i>Current Protocols</i> is issuing corrections for the following protocol article.</p><p>Reuven, O., Mikula, I., Ashkenazi-Preiser, H., Twaik, N., Ben-Meir, K., Meirow, Y., Daniel, L., Kariv, G., Kurd, M., &amp; Baniyash, M. (2022). Functional assays evaluating immunosuppression mediated by myeloid-derived suppressor cells. <i>Current Protocols</i>, <i>2</i>, e557. doi: 10.1002/cpz1.557</p><p>In the above-referenced article:</p><p>In step 11 of Support Protocol 2, “0.03125 µg/ml” has been changed to “0.03125 mg/ml”.</p><p>The current version online now includes this correction and may be considered the authoritative version of record.</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 12","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70082","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142840597","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Establishing Immortalized Brown and White Preadipocyte Cell Lines from Young and Aged Mice","authors":"Xiangdong Wu,&nbsp;Salaheldeen Elsaid,&nbsp;Florian Levet,&nbsp;Winson Li,&nbsp;Sui Seng Tee","doi":"10.1002/cpz1.70072","DOIUrl":"10.1002/cpz1.70072","url":null,"abstract":"<p>Studying adipogenesis and adipocyte biology requires the isolation of primary preadipocytes from adipose tissues. However, primary preadipocytes have a limited lifespan, can only undergo a finite number of divisions, and often lose their original biological characteristics before becoming senescent. The repeated isolation of fresh preadipocytes, particularly from young pups or aged animals, is costly and time consuming. Immortalization of these cells offers a solution by overcoming cellular senescence and maintaining proliferative capacity, allowing for long-term studies without the continuous need to isolate new cells from animals. Immortalized cell lines thus provide a consistent and reproducible experimental model, significantly reducing variability across different animals. However, successfully establishing immortalized preadipocyte cell lines presents challenges, including selecting appropriate adipose tissue depots, isolating primary preadipocytes, and choosing an effective immortalization strategy. In this article, we present optimized protocols and share first-hand experiences establishing immortalized brown and white preadipocyte cell lines from young and aging mice. These protocols offer a valuable resource for researchers studying adipogenesis and metabolism. © 2024 Wiley Periodicals LLC.</p><p><b>Support Protocol 1</b>: Retrovirus production</p><p><b>Basic Protocol 1</b>: Isolation and culture of primary brown and white preadipocytes from mouse interscapular brown adipose tissue (iBAT) and subcutaneous white adipose tissue (sWAT) in the same region</p><p><b>Basic Protocol 2</b>: Immortalization of mouse brown and white preadipocytes</p><p><b>Basic Protocol 3</b>: Selection of immortalized preadipocytes</p><p><b>Basic Protocol 4</b>: Selection of single-cell clones of immortalized mouse preadipocytes</p><p><b>Basic Protocol 5</b>: Single-cell sorting in a 96-well plate using a flow cytometer for the selection of single-cell clones of immortalized preadipocytes</p><p><b>Support Protocol 2</b>: Cryopreservation of immortalized mouse preadipocytes</p><p><b>Support Protocol 3</b>: Thawing and culture of cryopreserved immortalized mouse preadipocytes</p><p><b>Support Protocol 4</b>: Subculture and expansion of immortalized mouse preadipocytes</p><p><b>Basic Protocol 6</b>: Differentiation of immortalized mouse brown and white preadipocytes</p><p><b>Support Protocol 5</b>: Identification of differentiated white and brown adipocytes</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 12","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-12-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142820430","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"In Vivo Bioluminescence Imaging of Tumor Progression in the Lewis Lung Carcinoma Orthotopic Mouse Model: A Comparison Between the Tail Vein Injection and Intranasal Instillation Methods","authors":"Miki Yamada-Hara,&nbsp;Naoki Takahashi,&nbsp;Ji Won Byun,&nbsp;Liping Zeng,&nbsp;Zhihe Wang,&nbsp;Arisachi Tanaka,&nbsp;Zahra Malakoutikhah,&nbsp;Tomoko Hayashi,&nbsp;Nicholas J. G. Webster,&nbsp;Eyal Raz,&nbsp;Samuel Bertin","doi":"10.1002/cpz1.70071","DOIUrl":"10.1002/cpz1.70071","url":null,"abstract":"<p>Metastasis remains a leading cause of cancer-related mortality, yet its study has been constrained by the lack of reliable animal models that faithfully replicate this complex process. Syngeneic models for studying lung cancer metastasis are limited, with the Lewis lung carcinoma (LLC) model being the most commonly employed. The conventional LLC orthotopic model involves injecting LLC cells intravenously (i.v.) via the tail vein into syngeneic C57BL/6 mice. However, this model has significant drawbacks, such as tumor development in multiple anatomical sites, incomplete lung tumor penetrance, and challenges in monitoring lung tumor growth. This article highlights the advantages of using luciferase-expressing LLC cells combined with bioluminescence imaging (BLI) to quantify tumor progression in live animals. We demonstrate that both white- and black-furred C57BL/6 mice can be used for BLI. Finally, we propose that intranasal (i.n.) instillation of LLC cells offers a valuable alternative to the traditional i.v. tail vein injection method, particularly for its simplicity and improved reproducibility. Although the LLC i.n. model does not recapitulate the metastasis process via the blood vascular route, it is an effective model for studying tumor seeding within the lungs and is particularly useful for analyzing the impact of the lung microenvironment on tumor initiation and progression. © 2024 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Lewis lung carcinoma intravenous injection method</p><p><b>Support Protocol</b>: In vivo bioluminescence imaging</p><p><b>Basic Protocol 2</b>: Lewis lung carcinoma intranasal instillation method</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 12","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-12-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142808886","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}