Methods in cell biology最新文献_第6页

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

Establishing Tyro3, Axl, and Mertk Chinese hamster ovary (CHO) reporter cell lines for cancer immunology and therapeutic applications. 建立Tyro3、Axl和Mertk中国仓鼠卵巢（CHO）报告细胞系用于肿瘤免疫和治疗应用。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-12-14 DOI: 10.1016/bs.mcb.2024.11.001

Ahmed Aquib, Ziren Wang, Varsha Gadiyar, Rachael Pulica, Christopher Varsanyi, Trevor Frederick, Wen-I Tsou, Stanley G Kimani, Sergey Smirnov, Mariana S De Lorenzo, Sergei V Kotenko, Raymond B Birge

{"title":"Establishing Tyro3, Axl, and Mertk Chinese hamster ovary (CHO) reporter cell lines for cancer immunology and therapeutic applications.","authors":"Ahmed Aquib, Ziren Wang, Varsha Gadiyar, Rachael Pulica, Christopher Varsanyi, Trevor Frederick, Wen-I Tsou, Stanley G Kimani, Sergey Smirnov, Mariana S De Lorenzo, Sergei V Kotenko, Raymond B Birge","doi":"10.1016/bs.mcb.2024.11.001","DOIUrl":"https://doi.org/10.1016/bs.mcb.2024.11.001","url":null,"abstract":"Tyro-3, Axl, and Mertk (abbreviated TAM receptors or TAMs), have well established functions in efferocytosis, the process by which apoptotic cells are engulfed by phagocytic cells such as macrophages and dendritic cells. In addition to their roles in efferocytosis, TAMs are also pleiotropic immune modulators that dampen inflammation and promote immune resolution and tolerance. Mice lacking one or more of the TAM receptors in various murine models leads to chronic inflammation and in some cases autoimmunity and chronic disease. In recent years, TAMs have emerged as important contributors in cancer, functioning both as oncogenic tyrosine kinases as well as immune modulators. Many recent studies indicate that TAM inhibitors, including monoclonal antibodies, kinase inhibitors, decoy receptors and ligands, and small molecular wedge inhibitors have therapeutic potential in cancer biology and immunotherapy. Here, we report the development and characterization of two type of TAM reporter lines that can be adapted to screen a wide range of TAM inhibitor types. The first involves TAM-IFN-γR1 chimeric CHO lines, where the extracellular domain of human TAM receptors is fused with the transmembrane and intracellular signaling domains of the human IFN-γ receptor chain. The second type of TAM reporter line described is the EGFR-TAM chimeric CHO lines, which involves fusing the extracellular domain of the EGFR receptor with the transmembrane and intracellular tyrosine kinase domains and cytosolic tail of TAM receptors. With minimal adaptation, both lines can be adopted for high throughput screening with immune-oncology applications.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"196 ","pages":"17-41"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144667965","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 of tumor interstitial fluid for metabolic determinations. 分离肿瘤间质液用于代谢测定。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-12-14 DOI: 10.1016/bs.mcb.2024.11.004

Ainhoa Ruiz-Iglesias, Ángel García-Aldea, Elena Nonnast, Rosa M Peregil, Santos Mañes

{"title":"Isolation of tumor interstitial fluid for metabolic determinations.","authors":"Ainhoa Ruiz-Iglesias, Ángel García-Aldea, Elena Nonnast, Rosa M Peregil, Santos Mañes","doi":"10.1016/bs.mcb.2024.11.004","DOIUrl":"https://doi.org/10.1016/bs.mcb.2024.11.004","url":null,"abstract":"Neoplastic cells are characterized by alterations in metabolic pathways, typically leading to an aberrant use of glycolysis even under aerobic conditions - a phenomenon known as the Warburg effect. One consequence of this metabolic shift is the production of lactate, an oncometabolite often found at elevated levels in tumors. Lactate not only fuels the growth of cancer cells but also promotes angiogenesis, immune escape, and metastasis, thereby contributing to tumor progression and resistance to therapy. This highlights the importance of lactate in cancer metabolism and underscores the need for methods to measure it. In this study, we describe various centrifugation and elution protocols to isolate interstitial fluid and measure lactate in experimental tumors. These tumors were generated in immunocompetent mice using the MC38 colon cancer cell line. We propose that, with minor modifications, the methods here described could be successfully adapted for use with tumors originating from other human or murine cell lines. Furthermore, these methods could potentially enable the detection of other oncometabolites in the tumor microenvironment, which could have significant implications for both basic research and therapeutic strategies.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"196 ","pages":"177-192"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144667971","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 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

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