Current Protocols in Cell Biology最新文献_第9页

A Streamlined Sample Preparation Method for Mass Spectrometric Analysis 质谱分析的流线型样品制备方法

Current Protocols in Cell Biology Pub Date : 2018-04-09 DOI: 10.1002/cpcb.40

Yun Xiong, Ying Zhang, Jun Yao, Guoquan Yan, Haojie Lu

{"title":"A Streamlined Sample Preparation Method for Mass Spectrometric Analysis","authors":"Yun Xiong, Ying Zhang, Jun Yao, Guoquan Yan, Haojie Lu","doi":"10.1002/cpcb.40","DOIUrl":"10.1002/cpcb.40","url":null,"abstract":"Mass spectrometry-based proteomic technology experienced remarkable advancement in the past decades. However, their application was still hampered by the complexity of sample preparation. Conventional strategies for sample preparation incorporate multiple time-consuming steps, including cell lysis, protein extraction, protease cleavage, and desalting. Thus, we explored a simplified method (the cell-absorb method) during which living cells were absorbed into vacuum-dried polyacrylamide gel and directly digested in gel into peptides for subsequent LC-MS/MS analysis. As a consequence, both of the steps for cell lysis and protein extraction involved in traditional protocol were skipped. In addition to the decrease in time, more proteins were identified. Indeed, 3022 proteins were identified by the cell-absorb method. Meanwhile, only 2642 and 2420 proteins were identified by the classical SDS-PAGE based method and the reported gel absorption-based method, respectively. The cell-absorb method exhibited apparent advantage in terms of the depth of proteome coverage. Furthermore, the number of proteins identified show excellent reproducibility with a CV (coefficient of variation) of 0.03 among three replicates using the cell-absorb method. These advantages suggest that cell-absorb method is a promising choice for mapping the whole proteome of cells. © 2018 by John Wiley & Sons, Inc.","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"78 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.40","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36339899","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 1

Probing Endoplasmic Reticulum Dynamics using Fluorescence Imaging and Photobleaching Techniques 利用荧光成像和光漂白技术探测内质网动力学

Current Protocols in Cell Biology Pub Date : 2018-02-15 DOI: 10.1002/0471143030.cb2107s60

Lindsey Costantini, Erik Snapp

{"title":"Probing Endoplasmic Reticulum Dynamics using Fluorescence Imaging and Photobleaching Techniques","authors":"Lindsey Costantini, Erik Snapp","doi":"10.1002/0471143030.cb2107s60","DOIUrl":"10.1002/0471143030.cb2107s60","url":null,"abstract":"This unit describes approaches and tools for studying the dynamics and organization of endoplasmic reticulum (ER) membranes and proteins in living cells using fluorescence microscopy. The ER plays a key role in secretory protein biogenesis, calcium regulation, and lipid synthesis. However, study of these processes has often been restricted to biochemical assays that average millions of lysed cells or imaging of static fixed cells. With new fluorescent protein (FP) reporter tools, sensitive commercial microscopes, and photobleaching techniques, investigators can interrogate the behaviors of ER proteins, membranes, and stress pathways in single live cells. Solutions are described for imaging challenges relevant to the ER, including the mobility of ER membranes, a range of ER structures, and the influence of post-translational modifications on FP reporters. Considerations for performing photobleaching assays for ER proteins are discussed. Finally, reporters and drugs for studying misfolded secretory protein stress and the unfolded protein response are described. Curr. Protoc. Cell Biol. 60:21.7.1-21.7.29. © 2013 by John Wiley & Sons, Inc.","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"60 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-02-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/0471143030.cb2107s60","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"32103714","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 18

In Vitro Dissection of Autophagy 自噬的体外解剖

Current Protocols in Cell Biology Pub Date : 2018-02-13 DOI: 10.1002/cpcb.33

Min Zhang, Dawei Liu, Liang Ge

引用次数: 2

Microfluidics-Assisted TIRF Imaging to Study Single Actin Filament Dynamics 微流体辅助TIRF成像研究单个肌动蛋白丝动力学

Current Protocols in Cell Biology Pub Date : 2018-02-13 DOI: 10.1002/cpcb.31

Shashank Shekhar

引用次数: 15

High Purity Isolation and Sensitive Quantification of Extracellular Vesicles Using Affinity to TIM4 细胞外囊泡的高纯度分离及对TIM4亲和力的灵敏定量分析

Current Protocols in Cell Biology Pub Date : 2018-02-13 DOI: 10.1002/cpcb.32

Takeshi Yoshida, Takamasa Ishidome, Rikinari Hanayama

引用次数: 28

Combining Fluorescence and Bioluminescence Microscopy to Study the Series of Events from Cellular Signal Transduction to Gene Expression 结合荧光显微镜和生物发光显微镜研究细胞信号转导到基因表达的一系列事件

Current Protocols in Cell Biology Pub Date : 2018-02-13 DOI: 10.1002/cpcb.35

Kazuhito Goda, Takeo Takahashi, Hirobumi Suzuki

引用次数: 1

Fluorescence Lifetime Imaging of a Caspase-3 Apoptosis Reporter Caspase-3凋亡报告基因的荧光寿命成像

Current Protocols in Cell Biology Pub Date : 2018-02-13 DOI: 10.1002/cpcb.36

Johanna M. Buschhaus, Anne E. Gibbons, Kathryn E. Luker, Gary D. Luker

引用次数: 11

Live-Cell Visualization of Calcium Flux in Vibratome-Cut Thick Sections of Viable Tumor Tissue 活体肿瘤组织振动原子切割厚切片中钙通量的活细胞可视化

Current Protocols in Cell Biology Pub Date : 2018-02-13 DOI: 10.1002/cpcb.37

James Koh, Joyce A. Hogue, Julie A. Sosa

{"title":"Live-Cell Visualization of Calcium Flux in Vibratome-Cut Thick Sections of Viable Tumor Tissue","authors":"James Koh, Joyce A. Hogue, Julie A. Sosa","doi":"10.1002/cpcb.37","DOIUrl":"10.1002/cpcb.37","url":null,"abstract":"This unit outlines a live-cell imaging approach developed for visualization of intracellular calcium flux in human parathyroid tumors following stimulation of the calcium-sensing receptor (CASR), a class C G protein–coupled receptor (GPCR). The primary assay readout, intracellular calcium release induced by activation of the inositol triphosphate receptor, is potentially generalizable to multiple other GPCR signaling events that utilize this common downstream signal transduction pathway. Advantages of the approach include: (1) preservation of native tissue context and positional information, allowing direct visualization of intratumoral functional heterogeneity; (2) quantitative documentation of reactivity to a physiological stimulus in an experimentally tractable ex vivo system; and (3) generation of a dynamic, functional classifier of tumor biochemical behavior to augment static marker assessment. The technical steps are performed in three sequential phases: (1) viable tissue sectioning; (2) fluorophore loading and tissue immobilization; and (3) live-cell confocal microscopy. This versatile method provides a straightforward platform for functional characterization of human tumors. © 2017 by John Wiley & Sons, Inc.","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"77 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.37","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10850237","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 2

Simple and Rapid Tissue Clearing Method for Three-Dimensional Histology of the Pancreas 简单快速的胰腺三维组织清除方法

Current Protocols in Cell Biology Pub Date : 2018-02-13 DOI: 10.1002/cpcb.34

Hang Sheung Wong, Patrick Ka Kit Yeung, Hei Ming Lai, Karen Siu Ling Lam, Wu Wutian, Sookja Kim Chung

{"title":"Simple and Rapid Tissue Clearing Method for Three-Dimensional Histology of the Pancreas","authors":"Hang Sheung Wong, Patrick Ka Kit Yeung, Hei Ming Lai, Karen Siu Ling Lam, Wu Wutian, Sookja Kim Chung","doi":"10.1002/cpcb.34","DOIUrl":"10.1002/cpcb.34","url":null,"abstract":"Previously, high-resolution three-dimensional imaging of a whole and intact pancreas was not possible, since light is scattered when it passes through cell compartments with different refractive indices. CLARITY is one of the tissue clearing techniques that has yielded success with the central nervous system. To preserve tissue integrity after delipidation, conventional protocols embed tissue in an acrylamide-based hydrogel, which involves the use of specialized equipment. Recently, we determined that the hydrogel-embedding step could be simplified and replaced by passive tissue fixation in 4% paraformaldehyde (PFA). The whole procedure is less time-consuming and less error-prone, and can be completed within a week, compared to conventional CLARITY protocols that may take weeks to complete. Here, the detailed stepwise procedures involved in the simplified CLARITY workflow are applied to the pancreas of wild-type and gene-knockout 6-week old mice expressing green fluorescent protein (GFP) under the mouse insulin 1 promoter (MIP-GFP). This technique could facilitate high-resolution, three-dimensional imaging of pancreatic islets and comparison between different mouse genotypes under different disease and treatment conditions. © 2017 by John Wiley & Sons, Inc.","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"77 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.34","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35242360","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 5

Isolation of Lipid Droplets from Cells by Density Gradient Centrifugation 密度梯度离心分离细胞脂滴的研究

Current Protocols in Cell Biology Pub Date : 2018-02-13 DOI: 10.1002/cpcb.10

Dawn L. Brasaemle, Nathan E. Wolins

引用次数: 42