Benoit Stijlemans
  • Post-Doc, Vrije Universiteit Brussel
Research fields
  • Immunology
Eimeria vermiformis Infection Model of Murine Small Intestine
Authors:  Patrícia Figueiredo-Campos, Cristina Ferreira, Birte Blankenhaus and Marc Veldhoen, date: 12/20/2018, view: 5030, Q&A: 0
Eimeria vermiformis is a tissue specific, intracellular protozoan that infects the murine small intestinal epithelia, which has been widely used as a coccidian model to study mucosal immunology. This mouse infection model is valuable to investigate the mechanisms of host protection against primary and secondary infection in the small intestine. Here, we describe the generation of an E. vermiformis stock solution, preparation of sporulated E. vermiformis to infect mice and determination of oocysts burden. This protocol should help to establish a highly reproducible natural infection challenge model to study immunity in the small intestine. The information obtained from using this mouse model can reveal fundamental mechanisms of interaction between the pathogen and the immune response, e.g., provided by intraepithelial lymphocytes (IEL) at the basolateral site of epithelial cells but also a variety of other immune cell populations present in the gut.
Measurement of TLR4 and CD14 Receptor Endocytosis Using Flow Cytometry
Authors:  Michael S. Schappe and Bimal N. Desai, date: 07/20/2018, view: 9054, Q&A: 0
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 robust and quantitative method to measure LPS-triggered TLR4 and CD14 receptor endocytosis, one of the earliest events of LPS detection. Here, we describe a flow cytometry-based method that we used recently to study the role of the ion channel TRPM7 in TLR4 endocytosis (Schappe et al., 2018). The assay relies on stimulating the cells with LPS and measuring the cell surface levels of TLR4 (or CD14) at various time points using flow cytometry. Although we detail the method specifically for TLR4 and CD14 from murine bone marrow-derived macrophages, it can be readily adapted to evaluate receptor endocytosis in a variety of other signaling contexts.
In vitro Demonstration and Quantification of Neutrophil Extracellular Trap Formation
Authors:  Dongsheng Jiang, Mona Saffarzadeh and Karin Scharffetter-Kochanek, date: 07/05/2017, view: 15700, Q&A: 0
In the recent decade, neutrophil extracellular traps (NETs) have been identified and confirmed as a new anti-microbial weapon of neutrophils. In this protocol, we describe easy methods to demonstrate NET formation by immunofluorescence staining of extracellular chromatin fiber with anti-DNA/Histone H1 antibody and quantification of NETs by using a non-cell-permeable DNA specific dye Sytox orange.
Assay to Evaluate BAL Fluid Regulation of Fibroblast α-SMA Expression
Authors:  Jennifer L. Larson-Casey and A. Brent Carter, date: 11/20/2016, view: 8042, Q&A: 0
Because transforming growth factor-β (TGF-β1) induces differentiation of fibroblasts to myofibroblasts, we developed a protocol to evaluate alveolar macrophage-derived TGF-β1 regulation of lung fibroblast differentiation (Larson-Casey et al., 2016). The protocol evaluates the ability of mouse bronchoalveolar lavage (BAL) fluid to alter fibroblast differentiation. Fibroblast differentiation was measured by the expression of α-smooth muscle actin (α-SMA).
Ear Inflammation and Whole-mount Ear Staining
Authors:  Jia Tong Loh, Merry Gunawan and I-hsin Su, date: 10/20/2016, view: 10272, Q&A: 0
The recruitment of circulating neutrophils from the bloodstream to the site of inflammation represents one of the earliest events during an innate immune response. During this response, neutrophils tether and roll along the vessel walls before transmigrating across the endothelium into the interstitial space to exert their functions. Here, we describe a protocol for the staining of intravascular and tissue-localized neutrophils following contact sensitization of the skin with croton oil. Visualization of the neutrophilic distribution in skin provides for a better interpretation of the local immune response.
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