Methods in cell biology最新文献_第9页

In vitro screening methods of novel immune checkpoint inhibitors related to T cell infiltration and anti-PD-1 resistance. 与 T 细胞浸润和抗 PD-1 抗性有关的新型免疫检查点抑制剂的体外筛选方法。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2024-08-13 DOI: 10.1016/bs.mcb.2024.07.006

Zhuoying He, Xiuman Zhou, Youmei Xiao, Yanfeng Gao

{"title":"In vitro screening methods of novel immune checkpoint inhibitors related to T cell infiltration and anti-PD-1 resistance.","authors":"Zhuoying He, Xiuman Zhou, Youmei Xiao, Yanfeng Gao","doi":"10.1016/bs.mcb.2024.07.006","DOIUrl":"https://doi.org/10.1016/bs.mcb.2024.07.006","url":null,"abstract":"Immune checkpoint blockade-based cancer immunotherapy is an effective tool for cancer treatment. PD-1/PD-L1 blockade, however, is limited by a low response rate and adaptive resistance. A growing body of studies has shown that the high stromal content dense with extracellular matrix plays a significant role in immune checkpoint blockade resistance as well as T cell exclusion. In addition to physically obstructing immune cell infiltration, the extracellular matrix (ECM) may also interact with T cell receptors to indirectly impair their effector function and lead to anti-PD-1 resistance. Anti-PD-1 resistance may thus be overcome by rupturing the physical barrier related negative immune regulation, which may improve T cell infiltration and the efficacy of cancer immunotherapy. Here, we offer two straightforward methods based on flow cytometry and confocal microscopy to evaluate the effectiveness of an inhibitor targeting the novel \"stromal checkpoint\" DDR1/collagen, which aims to facilitate T cell migration and infiltration of tumor spheres by overcoming collagen barriers. With minor variations, the same method can be easily modified to test the inhibitors that target other immune checkpoints, and the extracellular matrix-associated drug targets that mediate anti-PD-1 resistance.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"190 ","pages":"11-24"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142623755","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

Myeloid-derived suppressor cells: Identification and function. 髓源性抑制细胞：识别与功能

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2024-09-10 DOI: 10.1016/bs.mcb.2024.07.009

Paola Vacca, Maria Teresa Bilotta, Lorenzo Moretta, Nicola Tumino

引用次数: 0

Measuring the impact of therapy-induced senescence on NK cell phenotypes in cancer. 测量治疗诱导的衰老对癌症中 NK 细胞表型的影响。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2024-08-23 DOI: 10.1016/bs.mcb.2024.07.010

Shreya R Chowdhury, Katherine C Murphy, Chaitanya N Parikh, Kelly D DeMarco, Lin Zhou, Marcus Ruscetti

{"title":"Measuring the impact of therapy-induced senescence on NK cell phenotypes in cancer.","authors":"Shreya R Chowdhury, Katherine C Murphy, Chaitanya N Parikh, Kelly D DeMarco, Lin Zhou, Marcus Ruscetti","doi":"10.1016/bs.mcb.2024.07.010","DOIUrl":"https://doi.org/10.1016/bs.mcb.2024.07.010","url":null,"abstract":"Cellular senescence is a damage-induced condition characterized by enduring cell cycle arrest and a heightened secretory profile known as the senescence-associated secretory phenotype (SASP). The SASP consists not only of release of inflammatory cytokines and chemokines that attract and activate a diverse repertoire of innate and adaptive immune cells, but also the upregulation of immunomodulatory cell surface molecules that promote immune clearance of senescent cells. Natural Killer (NK) cells are particularly adept at sensing and eliminating senescent cells. In the setting of cancer, commonly administered cytotoxic and cytostatic therapies can elicit senescence and in turn reactivate NK cell immune surveillance against tumors. Here, we detail a series of in vivo, ex vivo, and in vitro assays to assess the impact of therapy-induced senescence on NK cell phenotypes, including their activation, exhaustion, migration, and killing capacity in the context of pancreatic cancer. Importantly, this methodology can be adapted to investigate NK cell biology across various disease states and treatment modalities and help inform NK cell-based immunotherapies for cancer.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"190 ","pages":"171-201"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142623757","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

Imaging intracellular components in situ using super-resolution cryo-correlative light and electron microscopy. 利用超分辨率冷冻相关光镜和电子显微镜对细胞内成分进行原位成像。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2024-03-07 DOI: 10.1016/bs.mcb.2024.02.027

Mart G F Last, Lenard M Voortman, Thomas H Sharp

{"title":"Imaging intracellular components in situ using super-resolution cryo-correlative light and electron microscopy.","authors":"Mart G F Last, Lenard M Voortman, Thomas H Sharp","doi":"10.1016/bs.mcb.2024.02.027","DOIUrl":"https://doi.org/10.1016/bs.mcb.2024.02.027","url":null,"abstract":"Super-resolution cryo-correlative light and electron microscopy (SRcryoCLEM) is emerging as a powerful method to enable targeted in situ structural studies of biological samples. By combining the high specificity and localization accuracy of single-molecule localization microscopy (cryoSMLM) with the high resolution of cryo-electron tomography (cryoET), this method enables accurately targeted data acquisition and the observation and identification of biomolecules within their natural cellular context. Despite its potential, the adaptation of SRcryoCLEM has been hindered by the need for specialized equipment and expertise. In this chapter, we outline a workflow for cryoSMLM and cryoET-based SRcryoCLEM, and we demonstrate that, given the right tools, it is possible to incorporate cryoSMLM into an established cryoET workflow. Using Vimentin as an exemplary target of interest, we demonstrate all stages of an SRcryoCLEM experiment: performing cryoSMLM, targeting cryoET acquisition based on single-molecule localization maps, and correlation of cryoSMLM and cryoET datasets using scNodes, a software package dedicated to SRcryoCLEM. By showing how SRcryoCLEM enables the imaging of specific intracellular components in situ, we hope to facilitate adoption of the technique within the field of cryoEM.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"187 ","pages":"223-248"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140852362","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

Molecular cytometry for comprehensive immune profiling. 用于全面免疫分析的分子细胞仪。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2024-04-10 DOI: 10.1016/bs.mcb.2024.02.020

Pratip K Chattopadhyay

引用次数: 0

Measuring response to therapy in AML: Difference from normal flow cytometry vs RQ-PCR. 测量急性髓细胞性白血病的治疗反应：流式细胞术与 RQ-PCR 的差异。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2024-03-14 DOI: 10.1016/bs.mcb.2024.02.019

Michael R Loken, Chad A Hudson

引用次数: 0

Correlative cryo-microscopy pipelines for in situ cellular studies. 用于原位细胞研究的相关冷冻显微镜管道。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2024-03-27 DOI: 10.1016/bs.mcb.2024.02.038

Anna Pepe, Johannes Groen, Chiara Zurzolo, Anna Sartori-Rupp

{"title":"Correlative cryo-microscopy pipelines for in situ cellular studies.","authors":"Anna Pepe, Johannes Groen, Chiara Zurzolo, Anna Sartori-Rupp","doi":"10.1016/bs.mcb.2024.02.038","DOIUrl":"https://doi.org/10.1016/bs.mcb.2024.02.038","url":null,"abstract":"Correlative cryo-microscopy pipelines combining light and electron microscopy and tomography in cryogenic conditions (cryoCLEM) on the same sample are powerful methods for investigating the structure of specific cellular targets identified by a fluorescent tag within their unperturbed cellular environment. CryoCLEM approaches circumvent one of the inherent limitations of cryo EM, and specifically cryo electron tomography (cryoET), of identifying the imaged structures in the crowded 3D environment of cells. Whereas several cryoCLEM approaches are based on thinning the sample by cryo FIB milling, here we present detailed protocols of two alternative cryoCLEM approaches for in situ studies of adherent cells at the single-cell level without the need for such cryo-thinning. The first approach is a complete cryogenic pipeline in which both fluorescence and electronic imaging are performed on frozen-hydrated samples, the second is a hybrid cryoCLEM approach in which fluorescence imaging is performed at room temperature, followed by rapid freezing and subsequent cryoEM imaging. We provide a detailed description of the two methods we have employed for imaging fluorescently labeled cellular structures with thickness below 350-500nm, such as cell protrusions and organelles located in the peripheral areas of the cells.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"187 ","pages":"175-203"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140851996","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 telomerase activity using TRAP assays. 使用 TRAP 检测法测量端粒酶活性。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2023-05-16 DOI: 10.1016/bs.mcb.2022.12.009

Gabriele Saretzki

{"title":"Measuring telomerase activity using TRAP assays.","authors":"Gabriele Saretzki","doi":"10.1016/bs.mcb.2022.12.009","DOIUrl":"10.1016/bs.mcb.2022.12.009","url":null,"abstract":"Telomerase is a reverse transcriptase that consists of the telomerase reverse transcriptase (TERT) protein and the telomerase RNA component TERC which also harbors the template region for telomere synthesis. In its canonical function the enzyme adds single-stranded telomeric hexanucleotides de novo to the ends of linear chromosomes, telomeres, in telomerase-positive cells such as germline, stem- and cancer cells. This potential biochemical activity of telomerase can be measured with the help of a telomerase repeat amplification protocol (TRAP) which often includes a PCR amplification due to the low abundance of telomerase in most cells and tissues. The current chapter describes various TRAP methods to detect telomerase activity (TA) using gel-based methods, its advantages and deficits, how to perform an ELISA-based TRAP assay and how best to interpret its results. Since development of the TRAP assay in 1994, there have been numerous modifications and adaptations of the method from real-time PCR analysis, isothermal amplification and nanotechnology to CRISPR/Cas-based methods which will be briefly mentioned. However, it is not possible to cover all different TRAP methods and thus there is no comprehensiveness claimed by this chapter. Instead, the author describes various aspects of using TRAP assays including required controls, sample preparation, etc. in order to avoid pitfalls and set-backs in applying this rather complex and demanding technique. The TRAP assay is particularly important to support clinical diagnosis of cancer, analyze tumor therapy as well as to evaluate various approaches to inhibit TA as a form of anti-cancer therapy.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"181 ","pages":"127-149"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139672194","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

Adoptive immunotherapy with cells from tumor-draining lymph nodes activated and expanded in vitro. 利用在体外激活和扩增的肿瘤排泄淋巴结细胞进行采纳性免疫疗法。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2023-10-09 DOI: 10.1016/bs.mcb.2023.04.002

Carolyn Haynes, Laura Graham, Harry D Bear

{"title":"Adoptive immunotherapy with cells from tumor-draining lymph nodes activated and expanded in vitro.","authors":"Carolyn Haynes, Laura Graham, Harry D Bear","doi":"10.1016/bs.mcb.2023.04.002","DOIUrl":"10.1016/bs.mcb.2023.04.002","url":null,"abstract":"Tumor-draining lymph nodes (tumor-DLNs) provide a rich source of tumor-reactive lymphocytes which can be used in adoptive immunotherapy (AIT) and that circumvent the need to resect autologous tumor, without the challenges and shortcomings associated with using autologous tumor or anti-CD3 monoclonal antibody. Bryostatin/Ionomycin (Bryo/Io) provide a useful method of activating tumor-DLNs such that they can readily be expanded to sufficient numbers to be used in AIT, and growing the tumor-DLN lymphocytes in the gamma chain cytokines IL-7 plus IL-15 is superior to IL-2 in terms of T cell numbers and phenotype. AIT with these cells induces tumor regression and provides protection against metastases and future tumor challenge. Here, we provide a stepwise protocol to sensitize tumor-DLN cells in donor mice, activate tumor-DLN T cells ex vivo using Bryo/Io, expansion of these cells in gamma chain cytokines and adoptive transfer of the expanded cells back into tumor-bearing hosts. Methods relevant to these experiments, such as injecting tumor cells intravenously and monitoring for pulmonary metastases, tumor volume measurement and resection, and use of luciferase-expressing tumor cells to monitor for metastases following resection, are described in detail. The methods outlined herein can be easily adapted to suit similar experiments across multiple tumor cell lines and syngeneic mouse models.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"183 ","pages":"355-380"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140318581","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

In vitro re-challenge of CAR T cells. CAR T 细胞的体外再挑战。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2023-09-15 DOI: 10.1016/bs.mcb.2023.06.003

Clara Helena Klee, Alicia Villatoro, Nicholas Paul Casey, Else Marit Inderberg, Sébastien Wälchli

引用次数: 0