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

Large-scale CRISPR-Cas9 screens to define regulators of immune checkpoints. 大规模CRISPR-Cas9筛选确定免疫检查点的调节因子。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2025-11-20 DOI: 10.1016/bs.mcb.2025.10.011

Michael Mu, Johannes C Melms, Patricia Ho, Benjamin Izar

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":"https://doi.org/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

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

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":"https://doi.org/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

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":"https://doi.org/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

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":"https://doi.org/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

Measuring in vitro induction of antigen-specific CD8⁺ T-cell responses by murine conventional dendritic cells. 小鼠常规树突状细胞体外诱导抗原特异性CD8+ t细胞反应的测定。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2026-04-03 DOI: 10.1016/bs.mcb.2026.02.024

Mo D Staal, Douwe M T Bosma, Jannie Borst

引用次数: 0

Measurement and quantification of macrophage lytic cell death by kinetic microscopy. 巨噬细胞溶解细胞死亡的动力学显微镜测定和定量。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2026-03-25 DOI: 10.1016/bs.mcb.2026.02.019

Sara Cahill, Fiachra Humphries

{"title":"Measurement and quantification of macrophage lytic cell death by kinetic microscopy.","authors":"Sara Cahill, Fiachra Humphries","doi":"10.1016/bs.mcb.2026.02.019","DOIUrl":"https://doi.org/10.1016/bs.mcb.2026.02.019","url":null,"abstract":"Macrophages are innate immune cells that are critical in the maintenance of tissue homeostasis and defense against pathogens. Programmed cell death is a critical tool macrophages use to clear pathogens and to alert surrounding cells to damage following induction of cell death. Diverse forms of cell death, including pyroptosis, necroptosis, ferroptosis, and secondary apoptosis following apoptosis, result in loss of plasma membrane integrity. Membrane disruption occurs following the oligomerization of pore-forming proteins into the cell membrane, releasing cytokines and damage associated molecular patterns that trigger the immune response. Thus, quantification of cell membrane permeability is an effective method for assessing cell death in macrophages. Many different factors, including macrophage polarization, stimulation, and inflammatory state, can impact macrophage predisposition to, and rate of, programmed cell death. Thus, it is important to have a method of accurately assessing macrophage cell death kinetically, rather than at a single endpoint. In this protocol, we describe a protocol for assessing cell death in macrophages by quantifying cell membrane permeability using kinetic microscopy. This method overcomes limitations of common single time point metrics for assessing cell death and is adaptable and scalable for use in assessing cell death across different cell types and treatment conditions.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"206 ","pages":"109-122"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147817111","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