Methods in cell biology最新文献_第5页

Quantification of intracellular and mitochondrial ATP content in macrophages during lipopolysaccharide-induced inflammatory response. 脂多糖诱导炎症反应中巨噬细胞胞内和线粒体ATP含量的定量分析。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-02-26 DOI: 10.1016/bs.mcb.2024.01.006

Paulraj Kanmani, Guochang Hu

{"title":"Quantification of intracellular and mitochondrial ATP content in macrophages during lipopolysaccharide-induced inflammatory response.","authors":"Paulraj Kanmani, Guochang Hu","doi":"10.1016/bs.mcb.2024.01.006","DOIUrl":"10.1016/bs.mcb.2024.01.006","url":null,"abstract":"Sepsis, a condition characterized by systemic infection that becomes aggravated and dysregulated, is a significant cause of mortality in critically ill patients. Emerging evidence suggests that severe sepsis is often accompanied by alterations in cell metabolism, particularly mitochondrial dysfunction, resulting in multiorgan failure. Normally, metabolically active cells or tissues exhibit higher levels of mitochondrial turnover, respiration, and adenosine triphosphate (ATP) synthesis. However, during sepsis, these processes become overwhelmed or dysregulated, leading to impaired ATP production in mitochondria. Here, we present two straightforward protocols for quantifying ATP production from mitochondria in bone marrow-derived macrophages (BMDMs). Our workflow facilitates the easy isolation of BMDMs and mitochondria from BMDMs treated with lipopolysaccharide (LPS), the major cell wall component of Gram-negative bacteria. We quantified intracellular and mitochondrial ATP production in macrophages in vitro using this protocol. The results indicated a decrease in mitochondrial ATP content in BMDMs in response to LPS. With minimal adjustments, this method can be adapted for use with various human and mouse primary cells and cell lines.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"194 ","pages":"77-92"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143586247","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

Isolation and characterization of primary NK cells and the enrichment of the KIR2DL1⁺ population. 原代NK细胞的分离、鉴定及KIR2DL1+群体的富集。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-03-02 DOI: 10.1016/bs.mcb.2023.09.001

Batel Sabag, Abhishek Puthenveetil, Mira Barda-Saad

引用次数: 0

Efficient discrimination of functional hematopoietic stem cell progenitors for transplantation by combining alkaline phosphatase activity and CD34⁺ immunophenotyping. 结合碱性磷酸酶活性和CD34+免疫分型高效鉴别移植用功能性造血干细胞祖细胞。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2023-09-20 DOI: 10.1016/bs.mcb.2023.08.003

Laura G Rico, Jordi Juncà, Roser Salvia, Michael D Ward, Jolene A Bradford, Jordi Petriz

{"title":"Efficient discrimination of functional hematopoietic stem cell progenitors for transplantation by combining alkaline phosphatase activity and CD34+ immunophenotyping.","authors":"Laura G Rico, Jordi Juncà, Roser Salvia, Michael D Ward, Jolene A Bradford, Jordi Petriz","doi":"10.1016/bs.mcb.2023.08.003","DOIUrl":"10.1016/bs.mcb.2023.08.003","url":null,"abstract":"Alkaline phosphatase (ALP) is a membrane-associated hydrolase enzyme with dimeric structure that catalyzes phosphate esters, optimally at alkaline pH. ALP has a focus of interest, since this enzyme is highly expressed in primitive stem cells, such as progenitor cells, non-differentiating cells, and primordial cells. We previously adapted a fluorescent microscopy-based assay for quantifying ALPhigh and ALPlow cells by flow cytometry in combination with immunophenotyping. Our method uses a minimal sample perturbation approach, avoiding the use of erythrocyte lysing solutions and washing steps, and offering opportunities to combine live cell response and functional assessment with cell immunophenotyping, while minimizing sample preparation effects on the cell biology. Here we provide a detailed experiment protocol to determine alkaline phosphatase activity in CD34+ hematopoietic stem cells from blood and apheresis products obtained from patients involved in a stem cell mobilization process for allo- or auto-transplant. This study may provide the early detection of progenitors at different levels of differentiation and therefore, relate this information to long-term engraftment in hematopoietic stem cell transplants.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"195 ","pages":"101-113"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143780452","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

Multiparametric analysis of tumor infiltrating lymphocytes in solid tumors. 实体瘤浸润淋巴细胞的多参数分析。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2023-07-22 DOI: 10.1016/bs.mcb.2023.03.006

Rebecca Borella, Annamaria Paolini, Beatrice Aramini, Lara Gibellini, Valentina Masciale, Domenico Lo Tartaro, Massimo Dominici, Sara De Biasi, Andrea Cossarizza

引用次数: 0

PD-L1 expression in multiple myeloma myeloid derived suppressor cells. PD-L1在多发性骨髓瘤髓源性抑制细胞中的表达。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2025-01-17 DOI: 10.1016/bs.mcb.2024.11.006

Laura G Rico, Roser Salvia, Jolene A Bradford, Michael D Ward, Jordi Petriz

{"title":"PD-L1 expression in multiple myeloma myeloid derived suppressor cells.","authors":"Laura G Rico, Roser Salvia, Jolene A Bradford, Michael D Ward, Jordi Petriz","doi":"10.1016/bs.mcb.2024.11.006","DOIUrl":"10.1016/bs.mcb.2024.11.006","url":null,"abstract":"The Programmed Cell Death Protein 1/Programmed Cell Death Protein Ligand 1 (PD-1/PD-L1) axis stands as one of the most widely acknowledged targets for cancer immunotherapy. This ligand is considered a therapeutic target for this disease as it might play an important role in tumor immune evasion and drug resistance. In multiple myeloma (MM), PD-L1 is overexpressed in abnormal plasma cells and Myeloid-Derived Suppressor Cells (MDSCs). In MDSCs, unlike tumoral cells or derived cell lines, the PD-L1 protein is presented in a conformation not recognized by the monoclonal antibody. In contrast, when stimulating the sample with PMA, the PD-L1 molecule undergoes a conformational change that enables its recognition. Hence, we have developed a flow cytometric screening assay to determine PD-L1 conformational changes in MDSCs based on a minimal manipulation of the sample, to preserve the structure and functionality of the ligand. In this chapter, we provide detailed protocols to assess PD-L1 levels in MDSCs together with the representative results obtained in multiple myeloma patients. The obtained results enable the classification of MM patients based on the different PD-L1 detection after stimulation, which increases compared with unstimulated samples. We also provide protocols to assess kinetic analysis of PD-L1 expression over time and to compare PD-L1 cell surface expression with cytoplasmic expression. Finally, competitive experiments in the presence of durvalumab are also described to study its interaction with PD-L1. This approach can also be used to study the contribution of potential conformational changes in other proteins.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"195 ","pages":"115-141"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143780455","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

Optimizing protocols for human regulatory T isolation, expansion, and characterization. 优化人类调节性T的分离、扩增和表征方案。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-11-19 DOI: 10.1016/bs.mcb.2024.10.005

Sánchez-Moreno Inés, Martín-Otal Celia, Juan José Lasarte, Lozano Teresa

引用次数: 0

Induction of sepsis in a rat model by the cecal ligation and puncture technique. Application for the study of experimental acute renal failure. 盲肠结扎穿刺技术诱导大鼠脓毒症模型。应用于实验性急性肾功能衰竭的研究。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-06-12 DOI: 10.1016/bs.mcb.2024.05.004

María Ángeles González-Nicolás, Alberto Lázaro

引用次数: 0

Specific selection of stimulation-responsive γδ T-cells utilizing a short-term activation assay. 利用短期激活试验特异性选择刺激反应性γδ t细胞。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-11-14 DOI: 10.1016/bs.mcb.2024.10.006

Christian Peters, Jara Simeonov, Daniel Gombert, Dieter Kabelitz

{"title":"Specific selection of stimulation-responsive γδ T-cells utilizing a short-term activation assay.","authors":"Christian Peters, Jara Simeonov, Daniel Gombert, Dieter Kabelitz","doi":"10.1016/bs.mcb.2024.10.006","DOIUrl":"10.1016/bs.mcb.2024.10.006","url":null,"abstract":"T cells expressing the γδ T-cell receptor (TCR) represent a numerically small proportion of total T cells. Unlike αβ T cells they are activated by non-peptide antigens independently of MHC-presentation. γδ T cells have been recognized as a favorable prognostic marker across different tumor entities. Recently, γδ T cells (in particular Vδ2 T cells), have gained attention because of their effective intrinsic anti-tumor reactivity. Moreover, their ability for MHC-independent activation and in vitro expansion to high numbers makes them attractive candidates for tumor immunotherapy by adoptive transfer. In this regard, the ex vitro identification of highly reactive γδ T cells upon stimulation enables us to specifically identify, isolate and expand γδ T cells which potentially represent those with high anti-tumor reactivity. CD137 and CD154 represent suitable markers for identifying specifically activated γδ T cells. In humans, the surface mobilization of CD137 and CD154 reveals antigen-specific activation of regulatory (Treg) and conventional CD4 T cells, respectively. We adapted this method for the analysis of Vδ2 T cells, in which the mobilization of both CD137 and CD154 can be used to investigate their activation, whereby CD137 and CD154 do not discriminate regulatory from conventional cells. Thus, this method provides a new way to rapidly analyze quick changes in Vδ2 T-cell activation and allows for using these markers for cell sorting and subsequent expansion of the specifically reacting Vδ2 T cells.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"191 ","pages":"79-91"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143008169","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

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