Methods in cell biology最新文献_第4页

Simple protocol for measuring CD11b+ GR-1+ (Ly6C+/Ly6G+) myeloid cells from a minimum volume of mouse peripheral blood. 从最小体积的小鼠外周血中测量 CD11b+ GR-1+ (Ly6C+/Ly6G+)髓系细胞的简单方案。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2024-02-24 DOI: 10.1016/bs.mcb.2024.01.001

Eliana Borgna, Juan Cruz Gamba, Estefanía Prochetto, Iván Marcipar, Gabriel Cabrera

{"title":"Simple protocol for measuring CD11b+ GR-1+ (Ly6C+/Ly6G+) myeloid cells from a minimum volume of mouse peripheral blood.","authors":"Eliana Borgna, Juan Cruz Gamba, Estefanía Prochetto, Iván Marcipar, Gabriel Cabrera","doi":"10.1016/bs.mcb.2024.01.001","DOIUrl":"10.1016/bs.mcb.2024.01.001","url":null,"abstract":"Myeloid-derived suppressor cells (MDSCs) comprise a heterogeneous population of myeloid origin and immature state, whose hallmark is the capacity to suppress T cells and other immune populations. In mice, the first approach to identify MDSCs relies in the measurement of their phenotypical markers: CD11b and GR-1. In addition, two main subtypes of MDSCs have been defined based on the expression of the following markers: CD11b+ Ly6G- Ly6C+ (monocytic-MDSCs, M-MDSCs) and CD11b+ Ly6G+ Ly6C+/low (polymorphonuclear-MDSCs, PMN-MDSCs). Since CD11b+ GR-1+ (Ly6C+/Ly6G+) MDSCs can increase significantly in peripheral blood during numerous acute or chronic processes, measuring alterations in the phenotypic markers CD11b and GR-1 could be important as a first step before assessing the suppressive function of the cells. In many cases it could be necessary to measure CD11b+ Gr-1+ cells from a minimum volume of peripheral blood cells without greatly affecting animal viability, since this approach would allow for further studies to be conducted on subsequent days, such as measuring parameters of the immune response or even survival in the context of the pathology under study. The following protocol describes a simple and optimized protocol for measuring the presence of CD11b+ GR-1+ (Ly6C+/Ly6G+) myeloid cells using 2+ channel flow cytometry, from a minimum volume of mouse peripheral blood obtained by facial vein puncture.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140329979","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

Assessment of social behavior and chemosensory cue detection in an animal model of neurodegeneration. 评估神经变性动物模型的社交行为和化感线索检测。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2024-03-15 DOI: 10.1016/bs.mcb.2024.02.008

Adrián Portalés, Alberto Sánchez-Aguilera, Maria Royo, Sandra Jurado

引用次数: 0

Lateral fluid percussion injury: A rat model of experimental traumatic brain injury. 侧液叩击伤：实验性脑外伤大鼠模型

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2024-03-04 DOI: 10.1016/bs.mcb.2024.02.011

Saúl Huerta de la Cruz, Cindy Santiago-Castañeda, Erick J Rodríguez-Palma, Luisa Rocha, Maria Sancho

{"title":"Lateral fluid percussion injury: A rat model of experimental traumatic brain injury.","authors":"Saúl Huerta de la Cruz, Cindy Santiago-Castañeda, Erick J Rodríguez-Palma, Luisa Rocha, Maria Sancho","doi":"10.1016/bs.mcb.2024.02.011","DOIUrl":"10.1016/bs.mcb.2024.02.011","url":null,"abstract":"Traumatic brain injury (TBI) represents one of the leading causes of disability and death worldwide. The annual economic impact of TBI-including direct and indirect costs-is high, particularly impacting low- and middle-income countries. Despite extensive research, a comprehensive understanding of the primary and secondary TBI pathophysiology, followed by the development of promising therapeutic approaches, remains limited. These fundamental caveats in knowledge have motivated the development of various experimental models to explore the molecular mechanisms underpinning the pathogenesis of TBI. In this context, the Lateral Fluid Percussion Injury (LFPI) model produces a brain injury that mimics most of the neurological and systemic aspects observed in human TBI. Moreover, its high reproducibility makes the LFPI model one of the most widely used rodent-based TBI models. In this chapter, we provide a detailed surgical protocol of the LFPI model used to induce TBI in adult Wistar rats. We further highlight the neuroscore test as a valuable tool for the evaluation of TBI-induced sensorimotor consequences and their severity in rats. Lastly, we briefly summarize the current knowledge on the pathological aspects and functional outcomes observed in the LFPI-induced TBI model in rodents.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140331861","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

Correlative light and electron microscopy at defined cell cycle stages in a controlled environment. 在受控环境中，对确定的细胞周期阶段进行光镜和电子显微镜的相关观察。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2024-03-26 DOI: 10.1016/bs.mcb.2024.02.025

Helena Bragulat-Teixidor, Shotaro Otsuka

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":null,"pages":null},"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":null,"pages":null},"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":null,"pages":null},"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":null,"pages":null},"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