Methods in cell biology最新文献

A cell culture-based method for interrogating muscle to liver communication via secreted proteins. 一种基于细胞培养的方法，通过分泌蛋白质来询问肌肉与肝脏之间的通信。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2024-08-31 DOI: 10.1016/bs.mcb.2024.08.005

Ioannis Tsialtas, Kevin B Koronowski

{"title":"A cell culture-based method for interrogating muscle to liver communication via secreted proteins.","authors":"Ioannis Tsialtas, Kevin B Koronowski","doi":"10.1016/bs.mcb.2024.08.005","DOIUrl":"10.1016/bs.mcb.2024.08.005","url":null,"abstract":"Inter-organ communication, including the release of secreted proteins, plays a key role in synchronized physiological responses and organismal homeostasis. Recent studies have emphasized functions of muscle-secreted proteins (i.e., myokines), in regulating metabolic pathways and improving metabolic dysfunction distally in the liver. Thus, experimental workflows to study myokines and their impact on target cell types are of scientific value. Here, we describe a cell culture-based method to investigate communication from muscle to liver mediated by secreted proteins. Briefly, C2C12 myoblasts are differentiated into myotubes, myotube-conditioned media is collected, and myotube-secreted proteins are isolated and stored. To demonstrate the utility of this method, AML12 hepatocytes were treated with myotube-secreted proteins and effects on bioenergetics were assessed. This method can be useful as a proof of principle tool, for mechanistic studies, or paired with proteomic or biochemical analyses to identify novel myokines. We also envision it is adaptable in terms of cell type, downstream application, and signaling direction.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"200 ","pages":"197-210"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12641138/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145587962","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

2D and 3D cellular screening models and AI guided analysis. 二维和三维细胞筛选模型和人工智能引导分析。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 DOI: 10.1016/S0091-679X(26)00104-4

Oliver Kepp, Guido Kroemer

引用次数: 0

Super-resolution imaging of cell death in Drosophila tissues via expansion and pan-expansion microscopy. 果蝇组织细胞死亡的超分辨率扩增和泛扩增显微镜成像。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2026-03-09 DOI: 10.1016/bs.mcb.2026.02.004

Alina Kolpakova, Keren Yacobi-Sharon, Eli Arama

{"title":"Super-resolution imaging of cell death in Drosophila tissues via expansion and pan-expansion microscopy.","authors":"Alina Kolpakova, Keren Yacobi-Sharon, Eli Arama","doi":"10.1016/bs.mcb.2026.02.004","DOIUrl":"10.1016/bs.mcb.2026.02.004","url":null,"abstract":"Cell death is essential for the survival and homeostasis of nearly all organisms. Programmed cell death (PCD) originally described the reproducible elimination of specific cells during metazoan development. Apoptosis, the best-studied form of PCD, is characterized by distinct morphological, cellular, and biochemical hallmarks. However, the identification of alternative cell death pathways has highlighted the need to clearly distinguish among them, especially when exploring new paradigms of developmental alternative cell death pathways. Furthermore, the discovery of numerous non-lethal functions of apoptotic caspases, collectively termed non-lethal caspase-dependent processes (CDPs), has underscored the importance of differentiating apoptosis from CDPs within tissues in vivo. Morphological analysis remains essential for differentiating death pathways and for determining whether cells are dying or surviving, yet such analyses traditionally relied on electron microscopy, which is labor-intensive, costly, and technically demanding. Expansion microscopy (ExM), a technique that physically enlarges biological samples isotropically, has transformed light microscopy by enabling super- and ultra-resolution imaging using standard fluorescence or confocal platforms. Here, we describe two ExM-based protocols optimized for, but not limited to, Drosophila tissues: ExM, achieving ∼4× expansion, and pan-ExM, yielding ∼12-16× expansion. ExM supports super-resolution imaging of immunofluorescent labels, whereas pan-ExM provides access to ultrastructural features, albeit with limited capacity for specific protein detection. We also compare apoptotic and non-apoptotic cell death morphologies before and after ExM and pan-ExM. Given the growing diversity of ExM strategies, this chapter offers a comprehensive introduction to these rapidly advancing methodologies.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"208 ","pages":"221-248"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148226627","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Quantification of cell viability by automated analysis of live cell imaging. 通过活细胞成像的自动分析来定量细胞活力。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2026-03-19 DOI: 10.1016/bs.mcb.2026.02.010

Jan C d'Engelbronner, Stefanie Rufli, W Wei-Lynn Wong

引用次数: 0

Characterization of myofiber degeneration by assessment of ER-stress biomarkers. 通过评估内质网应激生物标志物来表征肌纤维变性。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2026-03-25 DOI: 10.1016/bs.mcb.2026.02.003

Serafina Oliverio, Cecilia Pesaresi, Fiorella Colasuonno, Luca Occhigrossi, Mauro Piacentini, Federica Rossin, Federica Di Sano

{"title":"Characterization of myofiber degeneration by assessment of ER-stress biomarkers.","authors":"Serafina Oliverio, Cecilia Pesaresi, Fiorella Colasuonno, Luca Occhigrossi, Mauro Piacentini, Federica Rossin, Federica Di Sano","doi":"10.1016/bs.mcb.2026.02.003","DOIUrl":"10.1016/bs.mcb.2026.02.003","url":null,"abstract":"The sarco-endoplasmic reticulum (SR/ER) forms an extensive network within muscle fibers, playing a central role in calcium (Ca2 +) homeostasis, protein synthesis, folding, sorting and quality control. ER functional disturbance causes endoplasmic reticulum (ER) stress and leads to the accumulation of unfolded or misfolded proteins, thereby triggering the unfolded protein response (UPR) to alleviate cellular stress and re-establish homeostasis. ER stress can be triggered by defects in the balance between protein folding and degradation, altered calcium and lipid levels and changes in ER-mitochondria contacts. Accumulating evidence suggests that ER stress may play an important part in the pathogenesis of inflammatory myopathies and genetic muscle disorders characterized by myofiber degeneration and replacement with fibrotic tissue. In particular, myopathy could be due to chronic ER stress and a maladaptive response leading to defective muscle force and atrophy. Considering this evidence, the characterization of degenerated muscle fibers could be conducted through the analysis of proteins involved in endoplasmic reticulum stress. Here we propose a protocol, intended as a practical and reproducible workflow, for evaluating ER-stress associated proteins in muscle tissue.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"208 ","pages":"149-163"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148226457","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Extracellular matrix and morphology assessment method on live 3D spheroids of thyroid carcinoma. 甲状腺癌三维活体球体的细胞外基质及形态学评价方法。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2025-09-27 DOI: 10.1016/bs.mcb.2025.09.011

Mario Udinotti, Udo Siebolts, Christoforos Vaxevanis, Barbara Seliger

{"title":"Extracellular matrix and morphology assessment method on live 3D spheroids of thyroid carcinoma.","authors":"Mario Udinotti, Udo Siebolts, Christoforos Vaxevanis, Barbara Seliger","doi":"10.1016/bs.mcb.2025.09.011","DOIUrl":"10.1016/bs.mcb.2025.09.011","url":null,"abstract":"In vitro tumor models might be useful to study tumor growth, invasion and therapy resistance, but also extracellular matrix (ECM) remodeling. This protocol will provide information for the generation of a 3D tumor cell cultures for high content analysis and describes a novel method to monitor the ECM morphology and remodeling as well as therapy responses using live 3D tumor cell spheroids. Using 3D spheroids of thyroid carcinoma (TC) cells as a model, our method involves the treatment of TC cells with inhibitory agents followed by subsequent analysis of ECM components to assess the influence of these drugs on the structural integrity of the EMT using fluorescence labeled antibodies (Ab) and confocal microscopy. Employing this method, the morphology of the formed spheroids under different conditions and co-cultures as well as the distribution of ECM components can be assessed, such as e.g. fibronectin 1 (FN1). The results will also provide valuable insights into the tumor microenvironment (TME) and potential interactions of viable spheroids with the components of the TME during the ECM remodeling process. The implementation of 3D spheroids for studying EMT in TC as a model has been shown to provide more accurate and representative results compared to traditional 2D monolayer cell cultures.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"201 ","pages":"169-184"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146097213","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Patient-derived models of tumor-immune cell interactions. 患者衍生的肿瘤免疫细胞相互作用模型。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2025-11-03 DOI: 10.1016/bs.mcb.2025.10.001

Niloofar Nemati, Nina Boeck, Zlatko Trajanoski

引用次数: 0

Fluorescence microscopic quantification of lysosomal cholesterol accumulation induced by cationic amphiphilic drugs. 阳离子两亲性药物致溶酶体胆固醇积累的荧光显微镜定量研究。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2025-10-06 DOI: 10.1016/bs.mcb.2025.09.009

Karla Alvarez-Valadez, Samy Dehissi, Lucille Ferret, Guido Kroemer, Mojgan Djavaheri-Mergny

{"title":"Fluorescence microscopic quantification of lysosomal cholesterol accumulation induced by cationic amphiphilic drugs.","authors":"Karla Alvarez-Valadez, Samy Dehissi, Lucille Ferret, Guido Kroemer, Mojgan Djavaheri-Mergny","doi":"10.1016/bs.mcb.2025.09.009","DOIUrl":"10.1016/bs.mcb.2025.09.009","url":null,"abstract":"Lysosomes are involved in the transport, degradation, and recycling of macromolecules through the autophagy and endocytosis pathways. Cholesterol is taken up by cells through the internalization of low-density lipoprotein (LDL) via LDL receptor-mediated endocytosis or micropinocytosis. Free cholesterol generated by the action of acid lipases contained in lysosomes can be transferred to other organelles. Dysfunctions in either cholesterol uptake or release from lysosomes can compromise the function and integrity of these organelles, thereby contributing to the pathogenesis of lysosomal storage disorders. We previously showed that some cationic amphiphilic drugs (CADs) mimic the phenotype of lysosomal storage disorders by inducing lysosomal cholesterol accumulation followed by lysosomal damage. Here, we describe two fluorescence microscopic methods for the visualization of cholesterol accumulation in lysosomes in response to the CAD leelamine. In the first method, the cell-permeable cholesterol analog labeled with the fluorophore BODIPY is used. In the second method, endogenous cholesterol-rich microdomains are labeled with filipin complex. Both methods imply the additional visualization of the lysosomal associated membrane protein 2 (LAMP2) by immunofluorescence. Finally, the role of lysosomal cholesterol accumulation in the induction of lysosomal membrane permeabilization (LMP) was assessed through a method based on the recruitment of Galectin-3 on damaged lysosomes.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"204 ","pages":"285-299"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147581868","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Quantitative assessment of nanoparticle uptake and trafficking in advanced 3D cell models: A high-content screening core to enhance precision nanomedicine development. 先进3D细胞模型中纳米颗粒摄取和运输的定量评估：高含量筛选核心，以提高精密纳米医学的发展。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2025-11-20 DOI: 10.1016/bs.mcb.2025.10.002

Meritxell B Cutrona

{"title":"Quantitative assessment of nanoparticle uptake and trafficking in advanced 3D cell models: A high-content screening core to enhance precision nanomedicine development.","authors":"Meritxell B Cutrona","doi":"10.1016/bs.mcb.2025.10.002","DOIUrl":"10.1016/bs.mcb.2025.10.002","url":null,"abstract":"Nanoparticles (NPs), the most common physical form of drug or therapeutic delivery systems enhance treatment safety and efficacy thanks to protection and vehiculation of their payload into the targeted tissue. Recently, nanomedicines have received increasing interest in the area of cancer therapeutics. However, the failure to overcome biological barriers at the tissue and cellular levels prevents NP distribution and delivery, which carries unsuccess in clinical trials. With the introduction of advanced three-dimensional (3D) tumor replicas such as cancer spheroids and patient-derived organoids (PDOs) that provide a physiological 3D tissue-like context, the intrinsic NP-cell interaction features such as cellular uptake and intratumor behavior can be directly tested on a translational and preclinical basis. High-content screening (HCS) plays a pivotal role for predicting the efficacy of each NP design in an automated and unbiassed manner. This chapter presents in detail some protocols developed for confocal microscopy HCS on miniaturized cultures of tumor spheroids and PDOs, quantitative profiling of NP intra-spheroid transport and RNA-interference screening to unveil NP penetrance cellular effectors. In perspective, these modules put together, envision a core for a nano-PDO HCS workflow to advance cancer nanomedicine development.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"204 ","pages":"245-284"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147581830","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

CRISPR-Cas9 knockout screens to identify drug resistance genes in acute myeloid leukemia. CRISPR-Cas9基因敲除筛选急性髓性白血病耐药基因

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2026-01-29 DOI: 10.1016/bs.mcb.2026.01.006

Xin Yang, Jiaqi Cai, Jiayi Wang, Yanyu Meng, Yufang Shi, Huan Cai

{"title":"CRISPR-Cas9 knockout screens to identify drug resistance genes in acute myeloid leukemia.","authors":"Xin Yang, Jiaqi Cai, Jiayi Wang, Yanyu Meng, Yufang Shi, Huan Cai","doi":"10.1016/bs.mcb.2026.01.006","DOIUrl":"10.1016/bs.mcb.2026.01.006","url":null,"abstract":"Acute Myeloid Leukemia (AML) is a hematopoietic malignancy characterized by the uncontrolled proliferation of aberrant myeloid blasts within the bone marrow, resulting in disrupted hematopoiesis and severe clinical consequences. Drug resistance represents a major barrier in AML treatment, frequently manifesting as relapse following initial remission with conventional chemotherapeutic agents such as cytarabine and venetoclax. The underlying mechanisms of drug resistance include enhanced drug efflux, altered drug metabolism, and activation of pro-survival signaling pathways, necessitating the elucidation of specific genetic determinants to enable the development of effective therapeutic strategies. The advent of CRISPR/Cas9 system has facilitated precise genomic modifications, permitting the generation of cell libraries with targeted gene knockouts in AML cells. This approach can identify genes whose disruption alters drug sensitivity, implicating their involvement in survival and resistance to cell death. This protocol outlines a systematic strategy to uncover genes associated with drug resistance in AML cells by leveraging CRISPR/Cas9-mediated functional genomic screening. By employing this methodology, genes conferring drug susceptibility upon knockout are noted as potential drivers of drug resistance, offering valuable insights for the rational design of targeted therapies.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"205 ","pages":"199-216"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147662954","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0