Mammalia

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    Protocols in Current Issue
    Bacterial Synchronized Transfer Assays in Bone Marrow Derived Macrophages
    Authors:  Sedelia R Dominguez, Tom Kawula and Shaun P Steele, date: 11/20/2019, view: 272, Q&A: 0
    [Abstract] Merocytophagy (“mero”, Greek for partial; “cytophagy” for cell eating) is a process by which cells acquire microbes and cytosolic material through phagocytosis of a small portion of neighboring cells upon cell-cell contact. Cell-cell contact dependent transfer events can be assessed through co-incubation of differently labeled cells. With these ...
    Isolation of Neutrophil Nuclei for Use in NETosis Assays
    Author:  Dave Boucher, date: 09/05/2019, view: 620, Q&A: 0
    [Abstract] Neutrophils are critical immune cells that protect our body against invading pathogens. They generate antibacterial DNA structures called neutrophil extracellular traps (NET). Recently we identified a new mechanism that enables NET formation. We observed that following recognition of lipopolysaccharides, inflammatory caspases cleave Gasdermin D ...
    Adoptive Transfer of Monocytes Sorted from Bone Marrow
    Authors:  Damya Laoui, Eva Van Overmeire, Chloé Abels, Jiri Keirsse and Jo A Van Ginderachter, date: 01/05/2019, view: 2081, Q&A: 0
    [Abstract] Inflammatory Ly6Chi monocytes can give rise to distinct mononuclear myeloid cells in the tumor microenvironment, such as monocytic myeloid-derived suppressor cells (Mo-MDSC), immature macrophages, M2-like tumor-associated macrophages (TAMs), M1-like TAMs or monocyte-derived dendritic cells (Mo-DCs). This protocol describes a method to ...
    Measurement of TLR4 and CD14 Receptor Endocytosis Using Flow Cytometry
    Authors:  Michael S. Schappe and Bimal N. Desai, date: 07/20/2018, view: 3760, Q&A: 0
    [Abstract] After recognizing extracellular bacterial lipopolysaccharide (LPS), the toll-like receptor 4 (TLR4)-CD14 signaling complex initiates two distinct signaling pathways–one from the plasma membrane and the other from the signaling endosomes (Kagan et al., 2008). Understanding the early stages of TLR4 signal transduction therefore requires a ...
    Quantification of Extracellular Double-stranded RNA Uptake and Subcellular Localization Using Flow Cytometry and Confocal Microscopy
    Authors:  Tan A Nguyen, Lachlan Whitehead and Ken C Pang, date: 06/20/2018, view: 2559, Q&A: 0
    [Abstract] Double-stranded RNA is a potent pathogen-associated molecular pattern (PAMP) produced as a by-product of viral replication and a well-known hallmark of viral infection. Viral dsRNAs can be released from infected cells into the extracellular space and internalized by neighboring cells via endocytosis. Mammals possess multiple pattern recognition ...
    Visualization of RNA at the Single Cell Level by Fluorescent in situ Hybridization Coupled to Flow Cytometry
    Authors:  Alice Coillard and Elodie Segura, date: 06/20/2018, view: 2951, Q&A: 0
    [Abstract] The protocol described here has been developed to detect RNA at the single cell level. Fluorescent probes hybridize to target RNAs and are detected by flow cytometry after multiple amplification steps. Different types of RNA can be detected such as mRNA, long noncoding RNA, viral RNA or telomere RNA and up to 4 different target probes can be used ...
    Ectopic Gene Expression in Macrophages Using in vitro Transcribed mRNA
    Authors:  Pallavi Chandra and Jennifer A. Philips, date: 05/20/2018, view: 2755, Q&A: 0
    [Abstract] Macrophages are immune cells that contribute to host defense through various mechanisms including phagocytosis and antigen presentation. Their antimicrobial capacity is subverted by clinically important intracellular pathogens such as Mycobacterium tuberculosis. The study of host-pathogen interactions using these cells is therefore of ...
    Generation of Luciferase-expressing Tumor Cell Lines
    Authors:  Todd V. Brennan, Liwen Lin, Xiaopei Huang and Yiping Yang, date: 04/20/2018, view: 8334, Q&A: 1
    [Abstract] Murine tumor models have been critical to advances in our knowledge of tumor physiology and for the development of effective tumor therapies. Essential to these studies is the ability to both track tumor development and quantify tumor burden in vivo. For this purpose, the introduction of genes that confer tumors with bioluminescent ...
    Mono Sodium Urate Crystal-induced Peritonitis for in vivo Assessment of Inflammasome Activation
    Authors:  Marianne R. Spalinger and Michael Scharl, date: 03/05/2018, view: 4013, Q&A: 0
    [Abstract] Due to its particulate material, mono-sodium urate (MSU) crystals are potent activators of the NOD-like receptor NLRP3. Upon activation, NLRP3 induces the formation of inflammasome complexes, which lead to the production and release of mature IL-1β. Bioactive IL-1β is a potent activator of innate immune responses and promotes recruitment of ...
    In vivo Leukemogenesis Model Using Retrovirus Transduction
    Authors:  Hiroshi Okuda and Akihiko Yokoyama, date: 12/05/2017, view: 4054, Q&A: 0
    [Abstract] Various genetic alterations such as chromosomal translocation cause leukemia. For examples, gene rearrangements of the mixed-lineage leukemia (MLL) gene generate MLL fusion genes, whose products are potent oncogenic drivers in acute leukemia. To better understand the mechanism of disease onset, several murine leukemia models ...



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