Methods in cell biology最新文献_第6页

Quantitative assessment of mitochondrial membrane potential in macrophages in sepsis.

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-02-29 DOI: 10.1016/bs.mcb.2024.01.004

Ajaz Ahmad, Paulraj Kanmani, Guochang Hu

{"title":"Quantitative assessment of mitochondrial membrane potential in macrophages in sepsis.","authors":"Ajaz Ahmad, Paulraj Kanmani, Guochang Hu","doi":"10.1016/bs.mcb.2024.01.004","DOIUrl":"https://doi.org/10.1016/bs.mcb.2024.01.004","url":null,"abstract":"Sepsis, a life-threatening condition characterized by dysregulated host response to infection, poses a significant public healthcare challenge. Excessive inflammatory responses during sepsis can lead to mitochondrial dysfunctions, resulting in organ damage. One hallmark of mitochondrial dysfunction is the reduction of mitochondrial membrane potential, which disrupts cellular metabolism, bioenergetics, and decreases the production of high-energy ATP through oxidative phosphorylation. In human sepsis, the mitochondrial membrane potential in peripheral blood monocytes has been identified as a marker of disease severity. Here, we present a detailed and widely accepted protocol for the detection of mitochondrial membrane potential using the JC-1 fluorescent dye in murine bone marrow-derived macrophages and J774A.1 macrophages following stimulation with lipopolysaccharides. This protocol is routinely employed and can be easily adapted for various cell types, intact tissues, and isolated mitochondria with minimal modifications. By utilizing this technique, researchers can gain valuable insights into mitochondrial function in different experimental contexts, potentially advancing our understanding of the pathogenesis and treatment of sepsis-related mitochondrial dysfunction.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"194 ","pages":"43-58"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143586251","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

Use of drug-killed cancer cells: A method to assess the therapeutic effectiveness of immunogenic cell death. 使用药物杀死的癌细胞：一种评估免疫原性细胞死亡治疗效果的方法。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-11-19 DOI: 10.1016/bs.mcb.2024.10.012

Kenny Misael Calvillo-Rodriguez, Maria Norma Gonzalez-Flores, Reyes Tamez-Guerra, Cristina Rodriguez-Padilla, Marilena Antunes-Ricardo, Ana Carolina Martinez-Torres

{"title":"Use of drug-killed cancer cells: A method to assess the therapeutic effectiveness of immunogenic cell death.","authors":"Kenny Misael Calvillo-Rodriguez, Maria Norma Gonzalez-Flores, Reyes Tamez-Guerra, Cristina Rodriguez-Padilla, Marilena Antunes-Ricardo, Ana Carolina Martinez-Torres","doi":"10.1016/bs.mcb.2024.10.012","DOIUrl":"https://doi.org/10.1016/bs.mcb.2024.10.012","url":null,"abstract":"Cancer immunotherapy has revolutionized cancer treatment by harnessing the immune system's potential to combat cancer. Among the various strategies in this field, the use of killed tumor cells (KC) induced by immunogenic cell death (ICD) inducers has gained attraction. This approach involves the treatment of cancer cells in vitro, followed by the subcutaneous injection of these killed cells into tumor-bearing mice. ICD induction triggers the exposure and release of damage-associated molecular patterns (DAMPs) and neoantigens, activating both innate and adaptive immune responses against cancer. Vaccination assays with immunocompetent mice and syngeneic cancer cells are considered the gold standard for identifying ICD inductors, as they effectively demonstrate the immunized host's capacity to achieve tumor rejection, typically showing more than 50% of protection. Despite significant progress in understanding ICD mechanisms, translating these findings into clinical practice faces challenges. Controversially, some reports indicate ICD induction with <50% protection in prophylactic vaccination. This variability in ICD interpretation can lead to \"false positives\" or overestimations of the immunogenicity of cell death induced by antitumor treatments, potentially complicating its clinical translation. Thus, rigorous adherence to the gold standard is necessary, and complementary experiments to assess the immunogenicity of cell death are advantageous. Here, we present a protocol to confirm the immunogenicity and therapeutic effectiveness of cell death induced by an ICD-inducer and evaluate its ability to reduce tumor burden in an established syngeneic mouse model.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"191 ","pages":"211-220"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143008186","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

Multidimensional profiling of cancer microenvironments in FFPE tissues by imaging mass cytometry. 肿瘤微环境在FFPE组织的多维谱成像细胞术。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-11-19 DOI: 10.1016/bs.mcb.2024.10.014

Marieke E Ijsselsteijn, Noel F C C de Miranda

引用次数: 0

Animal models of disease: Achievements and challenges.

4区生物学

Methods in cell biology Pub Date : 2025-01-01 DOI: 10.1016/S0091-679X(25)00026-3

José Manuel Bravo-San Pedro, Fernando Aranda, Aitziber Buqué, Lorenzo Galluzzi

引用次数: 0

Flow cytometry-based monitoring of myeloid-derived suppressor cells in the peripheral blood of patients with solid tumors. 基于流式细胞术的实体瘤患者外周血髓源性抑制细胞监测。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-11-13 DOI: 10.1016/bs.mcb.2024.10.009

Anna-Jasmina Donaubauer, Ilka Scheer, Rainer Fietkau, Udo S Gaipl, Benjamin Frey

{"title":"Flow cytometry-based monitoring of myeloid-derived suppressor cells in the peripheral blood of patients with solid tumors.","authors":"Anna-Jasmina Donaubauer, Ilka Scheer, Rainer Fietkau, Udo S Gaipl, Benjamin Frey","doi":"10.1016/bs.mcb.2024.10.009","DOIUrl":"https://doi.org/10.1016/bs.mcb.2024.10.009","url":null,"abstract":"Myeloid-derived suppressor cells (MDSCs) ameliorate inflammation by inhibiting T cell responses. In pathological conditions, such as autoimmunity, chronic infections or cancer they accumulate in the periphery. In cancer, MDSCs can also be part of the tumor microenvironment and are associated with a worse prognosis and limited response to immunotherapy. Nowadays attempts are made to specifically target MDSCs in cancer therapy. Still, the role of MDSCs in standard cancer treatment modalities, such as radiotherapy remains mostly elusive. Here, we describe a flow cytometry-based method to determine and monitor monocytic and granulocytic-derived MDSCs directly from whole blood in an easy, fast and reliable assay. As specific surface markers for MDSCs are lacking, the assay follows a gating strategy that excludes successively the main immune cells types and analyzes the remaining events for a set of molecules that are expressed on MDSCs. This assay is especially appropriate for longitudinal analyses and clinical trials and is suitable for being integrated into more complex immunophenotyping panels to generate a comprehensive immune status.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"191 ","pages":"135-150"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143008125","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 pre-clinical imaging of hypoxia and vascularity using MRI and PET. 定量临床前成像缺氧和血管的MRI和PET。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-11-19 DOI: 10.1016/bs.mcb.2024.10.016

Georgia Kanli, Selma Boudissa, Radovan Jirik, Tom Adamsen, Heidi Espedal, Hans Olav Rolfsnes, Frits Thorsen, Jesus Pacheco-Torres, Bassam Janji, Olivier Keunen

{"title":"Quantitative pre-clinical imaging of hypoxia and vascularity using MRI and PET.","authors":"Georgia Kanli, Selma Boudissa, Radovan Jirik, Tom Adamsen, Heidi Espedal, Hans Olav Rolfsnes, Frits Thorsen, Jesus Pacheco-Torres, Bassam Janji, Olivier Keunen","doi":"10.1016/bs.mcb.2024.10.016","DOIUrl":"https://doi.org/10.1016/bs.mcb.2024.10.016","url":null,"abstract":"During hypoxia, tissues are subjected to an inadequate oxygen supply, disrupting the balance needed to maintain normal function. This deficiency can occur due to reduced oxygen delivery caused by impaired blood flow or a decline in the blood's ability to carry oxygen. In tumors, hypoxia and vascularization play crucial roles, shaping their microenvironments and influencing cancer progression, response to treatment and metastatic potential. This chapter provides guidance on the use of non-invasive imaging methods including Positron Emission Tomography and Magnetic Resonance Imaging to study tumor oxygenation in pre-clinical settings. These imaging techniques offer valuable insights into tumor vascularity and oxygen levels, aiding in understanding tumor behavior and treatment effects. For example, PET imaging uses tracers such as [18F]-fluoromisonidazole (FMISO) to visualize hypoxic areas within tumors, while MRI complements this with anatomical and functional images. Although directly assessing tumor hypoxia with MRI remains challenging, techniques like Blood Oxygen Level Dependent (BOLD) and Dynamic Contrast-Enhanced MRI (DCE-MRI) provide valuable information. BOLD can track changes in oxygen levels during oxygen challenges, while DCE-MRI offers real-time access to perfusion and vessel permeability data. Integrating data from these imaging modalities can help assess oxygen supply, refine treatment strategies, enhance therapeutic effectiveness, and ultimately improve patient outcomes.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"191 ","pages":"289-328"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143008160","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

Single-cell RNA flow cytometry to assess intratumoral production of cytokines/chemokines. 单细胞RNA流式细胞术评估肿瘤内细胞因子/趋化因子的产生。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-11-20 DOI: 10.1016/bs.mcb.2024.10.013

Khiem C Lam, Romina S Goldszmid

引用次数: 0

Metallic nanoparticles biodistribution for the study of lymphoma in animal models.

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-08-23 DOI: 10.1016/bs.mcb.2024.07.004

Barbara Chalhoub, Víctor Franco Puntes, Laura Mondragón

{"title":"Metallic nanoparticles biodistribution for the study of lymphoma in animal models.","authors":"Barbara Chalhoub, Víctor Franco Puntes, Laura Mondragón","doi":"10.1016/bs.mcb.2024.07.004","DOIUrl":"https://doi.org/10.1016/bs.mcb.2024.07.004","url":null,"abstract":"T cell lymphoma constitutes a complex group of diseases, characterized by heterogeneous molecular features and clinical symptoms, and a dismal outcome no matter the therapeutic strategy chosen. In an attempt to improve patients' survival chances, treatment combinations (chemotherapy, radiotherapy, immunotherapy, gene therapy and thermotherapy) have been tested for their synergistic effects that may dramatically improve outcomes and reduce the side effects of each single modality treatment when therapeutic effects add up while side effects are distributed. In this context, nanoscale drug delivery agents have been developed and exploited to enhance the release of drugs in the treatment of several diseases, showing potential benefits in terms of pharmaceutical flexibility, selectivity, dose reduction and minimization of adverse effects. Inorganic materials (i.e., metal nanoparticles) can be used as imaging and radiotherapy agents demonstrating that nanoparticle-based therapies can combine and act as \"precision medicine\" for targeting tumors while leaving healthy tissue intact. Therefore, nanoparticles (NPs) appear as ideal platforms for multimodal therapy constituting a more than promising strategy in the search of effective combined treatments for T cell lymphoma. In our laboratory, we aim at validating these therapeutic strategies making use of metal NPs able to provide a diagnostic and therapeutic effect at the same time. Validation of the synthesized NPs will be possible thanks to the availability of an in vivo T cell lymphoma animal model also developed in the lab. Here, we describe basic protocols for the administration and biodistribution studies in solid tumors which could be of significant help for future therapies development and follow-up.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"192 ","pages":"159-180"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143039859","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

Multiplexed cytometry for single cell chemical biology.

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2023-07-14 DOI: 10.1016/bs.mcb.2023.03.007

Henry A M Schares, Madeline J Hayes, Joseph A Balsamo, Hannah L Thirman, Brian O Bachmann, Jonathan M Irish

{"title":"Multiplexed cytometry for single cell chemical biology.","authors":"Henry A M Schares, Madeline J Hayes, Joseph A Balsamo, Hannah L Thirman, Brian O Bachmann, Jonathan M Irish","doi":"10.1016/bs.mcb.2023.03.007","DOIUrl":"https://doi.org/10.1016/bs.mcb.2023.03.007","url":null,"abstract":"Flow cytometry has great potential for screening in translational research areas due to its deep quantification of cellular features, ability to collect millions of cells in minutes, and consistently expanding suite of validated antibodies that detect cell identity and functions. However, cytometry remains under-utilized in discovery chemical biology due to the differences in expertise between chemistry groups developing chemical libraries and cell biologists developing single cell assays. This chapter is designed to bridge this gap by providing a detailed protocol aimed at both chemistry and biology audiences with the goal of helping train novice researchers. Assay users select from three elements: a small molecule input, a target cell type, and a module of cytometry readouts. For each, we explore basic and advanced examples of inputs, including screening fractionated microbial extracts and pure compounds, and target cells, including primary human blood cells, mouse cells, and cancer cell lines. One such module of cytometry readouts focuses on cell function and measures DNA damage response (γH2AX), growth (phosphorylated S6), DNA content, apoptosis (cleaved Caspase3), cell cycle M phase (phosphorylated Histone H3), and viability (membrane permeabilization). The protocol can also be adapted to measure different functional readouts, such as cell identity or differentiation and contrasting cell injury mechanisms. The protocol is designed to be used in 96-well plate format with fluorescent cell barcoding and the debarcodeR algorithm. Ultimately, the goal is to encourage the next generation of chemical biologists to use functional cell-based cytometry assays in discovery and translational research.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"195 ","pages":"143-172"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143780454","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

Optimization and intra-assay validation of a multiparametric flow cytometric test for monitoring circulating TREGs. 用于监测循环 TREGs 的多参数流式细胞检测法的优化和测定内验证。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2024-07-09 DOI: 10.1016/bs.mcb.2024.06.005

Iole Macchia, Floriana Iacobone, Francesca Urbani

{"title":"Optimization and intra-assay validation of a multiparametric flow cytometric test for monitoring circulating TREGs.","authors":"Iole Macchia, Floriana Iacobone, Francesca Urbani","doi":"10.1016/bs.mcb.2024.06.005","DOIUrl":"https://doi.org/10.1016/bs.mcb.2024.06.005","url":null,"abstract":"Multiparametric flow cytometry (MFC) represents an essential tool for immune monitoring, and validation of MFC panels is a fundamental prerequisite in routine laboratory settings as well as for translational and clinical research purposes. Regulatory T cells (TREGs) constitute a subset of CD4+ effector T cells that modulate the immune response in numerous settings, including autoimmune disease, allergy, microbial infection, tumor immunity, transplantation, and more. These cells comprise a small fraction of total CD4+ T cells in human peripheral blood and mouse spleen. In oncology, TREG cells are highly relevant, as they are involved in the suppression of the anti-tumor response in many types of cancer, to the extent that the first immune checkpoint inhibitor approved for clinical use in humans was a monoclonal antibody directed against CTLA-4, a molecule functionally associated with TREGs. Due to all these factors, robust assays are mandatory to accurately determine TREG cell frequency and function. Here, we describe the validation of an 8-color flow-cytometry protocol for TREG detection and analysis in a real-world laboratory scenario. The entire process includes the workflow plan and the standard operating procedure resembling each phase, from the panel design to the staining, acquisition, and analysis steps. Validation is planned to be performed in replicates on fresh whole blood samples derived from multiple healthy subjects. The analytical validity of the TREG cell assay is ensured by testing the intra-assay accuracy. The detailed procedure for the entire process is accompanied by important troubleshooting suggestions and other useful tips.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"189 ","pages":"169-188"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142406543","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