编委
Emilie Besnard
  • Industry Scientist, Dorian Therapeutics
研究方向
  • Biochemistry, Cell Biology, Molecular Biology, Aging, Small Molecule
Proteome Birthdating: A Single-Sample Approach for Measuring Global Turnover Dynamics and “Protein Age”
蛋白质组生成年代测定:单样本解析整体蛋白质更新动态与“蛋白质年龄”方法

Within a cell, proteins have distinct and highly variable half-lives. As a result, the molecular ages of proteins can range from seconds to years. How the age of a protein influences its environmental interactions is a largely unexplored area of biology. To facilitate such studies, we recently developed a technique termed “proteome birthdating” that differentially labels proteins based on their time of synthesis. Proteome birthdating enables analyses of age distributions of the proteome by tandem mass spectrometry (LC–MS/MS) and provides a methodology for investigating the protein age selectivity of diverse cellular pathways. Proteome birthdating can also provide measurements of protein turnover kinetics from single, sequentially labeled samples. Here, we provide a practical guide for conducting proteome birthdating in in vitro model systems. The outlined workflow covers cell culture, isotopic labeling, protein extraction, enzymatic digestion, peptide cleanup, mass spectrometry, data processing, and theoretical considerations for interpretation of the resulting data.

Immunohistochemistry of Immune Cells and Cells Bound to in vivo Administered Antibodies in Liver, Lung, Pancreas, and Colon of B6/lpr Mice
B6/lpr小鼠肝脏、肺、胰腺和结肠中免疫细胞和与体内施用的抗体结合的细胞的免疫组织化学
作者:Kieran Adam and Adam Mor日期:07/20/2022,浏览量:2255,Q&A: 0

Employing a novel mouse model of immune related adverse events (irAEs) induced by combination of anti-PD1 and anti-CTLA-4 antibodies, we visualized immune infiltration into the liver, lung, pancreas, and colon. Here, we describe the avidin-biotin conjugate (ABC) method used to stain T cells (CD4 and CD8), B cells (CD19), macrophages (F4/80), and cells bound by the in vivo administered rat anti-mouse antibodies for chromogenic immunohistochemistry (IHC). Using a biotinylated goat anti-rat antibody, we detected the localization of cells bound to the in vivo antibodies for PD-1 and CTLA-4. IHC has advantages over other techniques, namely antibody availability, resistance to photobleaching, and greater sensitivity. Additionally, detection and localization of in vivo antibodies can be used in mice models to infer their therapeutic efficacy, stability, and function.


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Induction of Acute or Disseminating Bacterial Pneumonia in Mice and Sampling of Infected Organs for Studying the Host Response to Bacterial Pneumonia
小鼠急性或播散性细菌性肺炎的诱导和感染器官取样研究宿主对细菌性肺炎的反应
作者:Wanhai Qin, Zhe Liu, Tom van der Poll and Alex F. de Vos日期:01/05/2022,浏览量:3129,Q&A: 0

Experimental pneumonia models are important tools to study the pathophysiology of lung inflammation caused by microbial infections and the efficacy of (novel) drugs. We have applied a murine model of pneumonia induced by Pseudomonas (P.) aeruginosa infection to study acute host antibacterial defense in lungs, and assess epithelial cell specific responses as well as leukocyte recruitment to the alveolar space. To study host responses during disseminating pneumonia, we also applied a model of infecting mice with hypermucoviscous Klebsiella (K.) pneumoniae. In the latter model, K. pneumoniae is restricted to lung during the early phase of infection and at the later time points disseminates to the circulation and distal organs resulting in sepsis. Detailed procedures for induction of pneumonia in mice by Pseudomonas and Klebsiella and for isolation and analysis of infected organs, bronchoalveolar fluid, and bronchial brushes are provided in this article.


High-throughput Flow Cytometry Assay to Investigate TDP43 Splicing Function
高通量流式细胞术检测TDP43剪接功能
作者:H. Broder Schmidt and Rajat Rohatgi日期:04/20/2020,浏览量:4140,Q&A: 0
Mutations in RNA-binding proteins (RBPs) such as TDP43 are associated with transcriptome-wide splicing defects and cause severe neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The impact of RBP mutations on splicing function is routinely studied using PCR-based bulk measurements. However, the qualitative and low-throughput nature of this assay make quantitative and systematic analyses, as well as screening approaches, difficult to implement. To overcome this hurdle, we have developed a quantitative, high-throughput flow cytometry assay to investigate TDP43 splicing function on a single-cell level
Measuring Procaspase-8 and -10 Processing upon Apoptosis Induction
细胞凋亡诱导产生半胱天冬酶原-8和半胱天冬酶原-10的测定
作者:Sabine Pietkiewicz, Clara Wolfe, Jörn H. Buchbinder and Inna N. Lavrik日期:01/05/2017,浏览量:9320,Q&A: 0
Apoptosis or programmed cell death is important for multicellular organisms to keep cell homeostasis and for the clearance of mutated or infected cells. Apoptosis can be induced by intrinsic or extrinsic stimuli. The first event in extrinsic apoptosis is the formation of the Death-Inducing Signalling Complex (DISC), where the initiator caspases-8 and -10 are fully activated by several proteolytic cleavage steps and induce the caspase cascade leading to apoptotic cell death. Analysing the processing of procaspases-8 and -10 by Western blot is a commonly used method to study the induction of apoptosis by death receptor stimulation. To analyse procaspase-8 and -10 cleavage, cells are stimulated with a death ligand for different time intervals, lysed and subjected to Western blot analysis using anti-caspase-8 and anti-caspase-10 antibodies. This allows monitoring the caspase cleavage products and thereby induction of apoptosis.
Isolation of Intestinal Mesenchymal Cells from Adult Mice
从成年小鼠分离肠道间充质细胞
作者:Vasiliki Koliaraki and George Kollias日期:09/20/2016,浏览量:23295,Q&A: 0
During the last 20 years intestinal mesenchymal cells (IMCs) have emerged as an important cell type that plays a central role in intestinal development and homeostasis, by providing both structural support and growth regulatory elements. IMCs also actively participate in wound healing responses, thus regulating pathologic conditions such as tissue repair, inflammation, fibrosis and carcinogenesis (Powell et al., 2011). We have recently demonstrated that intestinal mesenchymal-specific signals play important in vivo physiological roles in intestinal inflammation and carcinogenesis (Koliaraki et al., 2012; Roulis et al., 2014; Koliaraki et al., 2015). Here we describe the enzymatic method used for the isolation and culture of mesenchymal cells from the adult mouse intestine.
Chromium-51 (51Cr) Release Assay to Assess Human T Cells for Functional Avidity and Tumor Cell Recognition
铬-51 (51Cr) 释放试验评估T淋巴细胞活性及对肿瘤细胞识别能力
作者:Petra Baumgaertner, Daniel E. Speiser, Pedro Romero, Nathalie Rufer and Michael Hebeisen日期:08/20/2016,浏览量:18212,Q&A: 0
Cytotoxic CD8+ T cells are able to specifically recognize and kill target cells through specific interaction between their T cell receptors (TCRs) and small immunogenic peptides (antigens) presented by major histocompatibility complex (MHC) molecules. The antigen recognition capacity and in vitro lytic activity of antigen-specific cytotoxic T cells can be assessed functionally in the so-called chromium 51 (51Cr) release assay, which was developed almost 50 years ago in our institution (Brunner et al., 1968). Radioactively-labelled cells deficient for endogenous antigen presentation [e.g., transporter for antigen presentation (TAP)-deficient T2 cells] and stably transfected with the MHC of interest (e.g., HLA-A2+) are typically used as targets during this 4h assay. Alternatively, 51Cr-labelled virus-infected or tumor cell lines presenting immunogenic antigens endogenously can serve as target cells (e.g., for the assessment of tumor recognition).

In a peptide titration assay (section A), radioactively labelled target cells are pulsed with a serial dilution of the antigenic peptide and incubated at an effector (e.g., a CD8+ T cell clone) to target (51Cr -T2 cells) ratio (E:T) of 10:1 in a 96-well V-bottom plate for 4 h at 37 °C. In a tumor killing assay (section B), cytotoxic CD8+ effector cells are incubated at different ratios with the 51Cr-labelled target cell line (typically at E:T ratios of 30:1, 10:1, 3:1 and 1:1) in the presence or absence of the specific antigenic peptide (1 μM) and incubated for 4 h at 37 °C. At the end of the test, the amount of radioactivity release from the lysed target cells is determined in the supernatant using a liquid scintillation counter. The percentage of specific lysis, as well as the EC50 (i.e., 50% of maximal killing) and EMax values are then calculated, providing quantitative information about the antigen-specific functional avidity (i.e., the relative efficiency of T cell function based on antigen recognition via a defined TCR and maximal killing capacity of the analyzed T cells).