Methods in cell biology最新文献_第7页

Flow cytometry conjugate formation assay between natural killer cells and their target cells. 流式细胞术测定自然杀伤细胞与靶细胞之间的偶联物形成。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-03-11 DOI: 10.1016/bs.mcb.2024.02.037

Gilles Iserentant, Carole Seguin-Devaux, Jacques Zimmer

{"title":"Flow cytometry conjugate formation assay between natural killer cells and their target cells.","authors":"Gilles Iserentant, Carole Seguin-Devaux, Jacques Zimmer","doi":"10.1016/bs.mcb.2024.02.037","DOIUrl":"10.1016/bs.mcb.2024.02.037","url":null,"abstract":"Before being able to kill other cells, natural killer (NK) cells first have to establish contact with those targets. In case of a predominance of activating signals from the target cell over inhibitory ones, the killing process is initiated. It is possible, with a simple two-color flow cytometry method, to evaluate, for any given effector cell-target cell pair, the number of conjugates between both types of cells. The percentage obtained gives an idea of the amplitude of binding of the NK cells to the targets and might be expected to be indicative of the level of cytotoxicity. Nevertheless, there is no absolute correlation, as the percentages of conjugates are sometimes higher with relatively resistant targets than with the highly sensitive cell line K562. Practically, NK cells and target cells are stained with two differently fluorescent dyes and incubated together at the desired effector:target ratio (in our example, 1:1) for various periods of time (0, 10, 30min, etc.) at 37°C. After the incubation time, the cells are carefully introduced into the flow cytometer, where in principle three populations are distinguished: the single positive, unconjugated effector and target cells, respectively, and the double positive subset, which corresponds to the conjugates between both cell types. We describe here in detail the staining and cell culture protocols and procedures, and give several examples. Thus, the very cytotoxic NK leukemia cell line KHYG-1 versus the myeloid leukemia K562 (the \"conventional\" NK cell target) and the Burkitt lymphoma cell line Raji forms a high number of conjugates. In contrast, purified, non-activated, healthy donor-derived peripheral blood NK cells bind less to the targets, in accordance with their low (K562) or absent (Raji) cytotoxic activity.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"193 ","pages":"213-228"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143370852","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

Image processing approaches for microtubule remodeling quantification at the immunological synapse. 免疫突触微管重构量化的图像处理方法。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-03-13 DOI: 10.1016/bs.mcb.2024.02.036

Daniel Krentzel, Maria Isabella Gariboldi, Marie Juzans, Marta Mastrogiovanni, Florian Mueller, Céline Cuche, Vincenzo Di Bartolo, Andrés Alcover

{"title":"Image processing approaches for microtubule remodeling quantification at the immunological synapse.","authors":"Daniel Krentzel, Maria Isabella Gariboldi, Marie Juzans, Marta Mastrogiovanni, Florian Mueller, Céline Cuche, Vincenzo Di Bartolo, Andrés Alcover","doi":"10.1016/bs.mcb.2024.02.036","DOIUrl":"10.1016/bs.mcb.2024.02.036","url":null,"abstract":"Immunological synapses result from a T cell polarization process, requiring cytoskeleton remodeling. Actin and microtubules drive synapse architecture and the localization of intracellular organelles, including Golgi and endolysosomal compartments, ensuring the directional localization of synapse components. Microtubule remodeling includes the centrosome polarization and the formation of a radial microtubules network, extending from the centrosome to the synapse periphery. Concomitantly, a ring of filamentous actin forms at the synapse periphery. Microtubule and actin remodeling facilitate vesicle fusion at the synapse, enabling T cell effector functions. Analyzing structural subtleties of cytoskeleton remodeling at the immunological synapse is crucial to understand its role in T cell functions. It may also pinpoint pathological states related with cytoskeletal dysfunctions. Quantifying filamentous protein network properties is challenging due to their complex and heterogeneous architectures and the inherent difficulty of segmenting individual filaments. Here, we describe the development of an image processing approach aimed at quantifying microtubule organization at the immunological synapse without the need for filament segmentation. The method is based on the analysis of the spatial and directional organization of microtubules growing from the centrosome to the synapse periphery. It is applied to investigate the importance of Adenomatous polyposis coli (Apc), a polarity regulator and tumor suppressor, in immunological synapse structure and functions and its potential implication in anti-tumor immune responses. We provide an open-source napari plugin of the outlined methods for analyzing filamentous networks.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"193 ","pages":"39-67"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143370855","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 mitochondrial membrane potential in macrophages in sepsis. 脓毒症中巨噬细胞线粒体膜电位的定量评价。

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":"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

Evaluating functional anti-tumor T cell responses in autologous 2D co-cultures using flow cytometry. 利用流式细胞术评估自体2D共培养的功能性抗肿瘤T细胞反应。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2025-01-31 DOI: 10.1016/bs.mcb.2025.01.002

Lucas Baldran-Groves, Jeroen Melief, Andreas Lundqvist

引用次数: 0

Animal models of disease: Achievements and challenges. 疾病的动物模型：成就与挑战。

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

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

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":"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

Organ-on-chip immunostaining method for three-dimensional identification and study of immune cells responding to drug-treated tumor cells. 器官芯片免疫染色法三维识别和研究免疫细胞对药物治疗肿瘤细胞的反应。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2025-02-05 DOI: 10.1016/bs.mcb.2025.01.005

Francesco Noto, Adele De Ninno, Mario Falchi, Luca Businaro, Giovanna Schiavoni, Fabrizio Mattei

{"title":"Organ-on-chip immunostaining method for three-dimensional identification and study of immune cells responding to drug-treated tumor cells.","authors":"Francesco Noto, Adele De Ninno, Mario Falchi, Luca Businaro, Giovanna Schiavoni, Fabrizio Mattei","doi":"10.1016/bs.mcb.2025.01.005","DOIUrl":"https://doi.org/10.1016/bs.mcb.2025.01.005","url":null,"abstract":"Epigenetic deregulation is implied in cancer initiation and resistance to antitumor drugs. In melanoma, aberrant DNA hypermethylation is frequently observed, resulting in the silencing of several genes involved in cell cycle regulation, apoptosis, tumor growth and drug resistance. DNA hypomethylating agents have been recently evaluated in both preclinical and clinical studies as a strategy to restore tumor suppressor genes and to increase immune recognition by tumors, highlighting their potential in pre-clinical models of melanoma. Advanced microfluidic system for the culture of complex three-dimensional cell, tissue and organ models have proven utility for oncoimmunology studies and drug testing. Here we present a protocol employing ad hoc fabricated microfluidic devices to reproduce a three-dimensional (3D) tumor microenvironment (TME) to study two aspects of the crosstalk between immune and cancerous cells under the effect of Decitabine (DAC), a DNA methyl transferase inhibitor (DNMTi). First, we evaluated the preferential migration of immune cells towards treated and non-treated melanoma cells inside the chip. Next, we identified a specific subpopulation of migrated immune cells, with an on-chip immunostaining protocol resulting in the acquisition and evaluation of 3D images on a Laser-Scanning Confocal Microscopy (LSCM) station for in-depth characterization of tumor-immune interactions. This protocol may find broad application for pre-clinical drug testing in cancer studies.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"196 ","pages":"209-223"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144667974","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":"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