| 1. |
- Amirbeagi, Firoozeh, et al.
(author)
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Determination of Subset-Restricted Anti-neutrophil Cytoplasmic Antibodies (ANCA) by Immunofluorescence Cytochemistry.
- 2019
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In: Methods in molecular biology (Clifton, N.J.). - New York, NY : Springer New York. - 1940-6029. ; , s. 63-77
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Book chapter (peer-reviewed)abstract
- Neutrophils have long been considered a homogeneous cell type where all circulating cells of a particular individual express the same proteins. Lately, however, this view is changing and distinct neutrophil subsets, defined by the presence or absence of different proteins, are being increasingly recognized. At least two separate protein markers, CD177 and Olfactomedin-4 (OLFM4) are known to be expressed by some, but not all, circulating neutrophils of a given individual. We recently described the existence of subset-restricted serum autoantibodies targeting OLFM4; these were discovered during clinical testing for anti-neutrophil cytoplasmic antibodies (ANCAs). ANCA testing is part of the clinical examinations routinely carried out to support diagnosis of suspected autoimmune conditions, especially vasculitis. Positive sera typically react with all neutrophils from a single donor, whereas subset-restricted ANCA sera (such as those containing anti-OLFM4 antibodies) only react with a fraction of neutrophils. Described in this chapter is an indirect immunofluorescence (IIF) approach to test human sera for the presence of subset-restricted ANCA as well as instructions for costaining experiments using sera and purified antibodies directed against established subset markers.
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| 2. |
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| 3. |
- Aufschnaiter, Andreas, Dr. rer. nat. 1988-, et al.
(author)
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Yeast Mitoribosome Purification and Analyses by Sucrose Density Centrifugation and Immunoprecipitation
- 2023
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In: Methods in Molecular Biology. - : Humana Press. - 1064-3745 .- 1940-6029. ; , s. 119-132, s. 119-132
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Book chapter (other academic/artistic)abstract
- Mitochondrial protein biosynthesis is maintained by an interplay between the mitochondrial ribosome (mitoribosome) and a large set of protein interaction partners. This interactome regulates a diverse set of functions, including mitochondrial gene expression, translation, protein quality control, and respiratory chain assembly. Hence, robust methods to biochemically and structurally analyze this molecular machinery are required to understand the sophisticated regulation of mitochondrial protein biosynthesis. In this chapter, we present detailed protocols for immunoprecipitation, sucrose cushions, and linear sucrose gradients to purify and analyze mitoribosomes and their interaction partners.
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| 4. |
- Bossios, Apostolos, 1969, et al.
(author)
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CD34+ eosinophil-lineage-committed cells in the mouse lung.
- 2014
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In: Methods in molecular biology (Clifton, N.J.). - New York : Springer New York. - 1940-6029. - 9781493910151 ; 1178, s. 29-43
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Book chapter (peer-reviewed)abstract
- Several studies suggest that eosinophil progenitor cells are capable of extramedullary proliferation but also enhance chronic inflammation via their own production of inflammatory and chemotactic mediators, thus augmenting the degree of inflammation by recruitment of more progenitors or mature effector cells, such as eosinophils at the site of inflammation. In this chapter, we provide methods focused on detecting eosinophil progenitor cells in the lung of allergen-challenged mice and how to monitor their proliferation capacity.
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| 5. |
- Brinkmalm, Ann, et al.
(author)
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Detection of α-Synuclein in Biological Samples Using Mass Spectrometry
- 2019
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In: Methods in molecular biology (Clifton, N.J.). - New York, NY : Springer. - 1940-6029. ; , s. 209-220
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Book chapter (peer-reviewed)abstract
- Here we describe a method using mass spectrometry to characterize and quantify immuno-enriched α-synuclein forms from biochemically fractionated brain tissue.
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| 6. |
- Bylund, Johan, 1975, et al.
(author)
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Measurement of respiratory burst products, released or retained, during activation of professional phagocytes.
- 2014
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In: Methods in molecular biology (Clifton, N.J.). - Totowa, NJ : Humana Press. - 1940-6029. ; 1124, s. 321-38
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Research review (peer-reviewed)abstract
- Activation of professional phagocytes, potent microbial killers of our innate immune system, is associated with an increase in cellular consumption of molecular oxygen (O2). The consumed O2 is utilized by an NADPH-oxidase to generate highly reactive oxygen species (ROS) by a one electron reduction, initially generating superoxide anion (O2 (-)) that then dismutates to hydrogen peroxide (H2O2). The ROS are strongly bactericidal molecules but may also cause tissue destruction, and are capable of driving immune competent cells of both the innate and the adaptive immune systems into apoptosis. The development of basic techniques to measure/quantify ROS generation by phagocytes during activation of the respiratory burst is of great importance, and a large number of methods have been used for this purpose. A selection of methods, including chemiluminescence amplified by luminol or isoluminol, the absorbance change following reduction of cytochrome c, and the fluorescence increase upon oxidation of PHPA, are described in detail in this chapter with special emphasis on how to distinguish between ROS that are released extracellularly, and those that are retained within intracellular organelles. These techniques can be valuable tools in research spanning from basic phagocyte biology to more clinically oriented research on innate immune mechanisms and inflammation.
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| 7. |
- Castegnaro, Filippo, 1994, et al.
(author)
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Preparation of Protein-Enriched Outer Membrane Vesicles from Escherichia Coli for In Situ Structural Biology of Outer Membrane Proteins
- 2023
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In: Methods in molecular biology (Clifton, N.J.). - 1940-6029. ; , s. 247-257
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Book chapter (peer-reviewed)abstract
- Bacterial outer membrane vesicles (OMVs) can be selectively enriched with one or more outer membrane proteins to allow the biophysical characterization of these membrane proteins embedded in the native cellular environment. Unlike reconstituted artificial membrane environments, OMVs maintain the native lipid composition as well as the lipid asymmetry of bacterial outer membranes. Here, we describe in detail the steps necessary to prepare OMVs, which contain high levels of a designated protein of interest, and which are of sufficient homogeneity and purity to perform biophysical characterizations using high-resolution methods such as atomic force microscopy, electron microscopy, or single-molecule force spectroscopy.
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| 8. |
- Christenson, Karin, et al.
(author)
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Analyzing cell death events in cultured leukocytes.
- 2012
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In: Methods in molecular biology (Clifton, N.J.). - Totowa, NJ : Humana Press. - 1940-6029. ; 844, s. 65-86
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Research review (peer-reviewed)abstract
- Cell death is of utmost importance in immunity, in part as a way to control the development and activity of leukocytes, but also as a strategy employed by leukocytes to rid the body of unwanted cells. Apoptosis is the classic type of programmed cell death involving an ordered sequence of cellular events, resulting in morphological changes that include cleavage/fragmentation of DNA, condensation of nuclei, cell shrinkage, and alterations of the plasma membrane. The apoptotic cell is a nonfunctional, but structurally intact, entity with preserved membrane integrity that is engulfed by surrounding cells (a process known as clearance) in an immunologically silent manner. In contrast, necrotic cells, i.e., nonfunctional cells that have lost membrane integrity, are freely permeable and leak intracellular constituents that may shift immunological homeostasis. Thus, membrane integrity of dead leukocytes is very important from an immunological point of view. For the analysis of leukocyte cell death, a wide variety of assays are available to monitor different events along the cell death pathway; a combination of different methods is advantageous in order to gain a more complete understanding of this dynamic process. In this chapter, we describe several in vitro methods for evaluating leukocyte cell death, mainly focusing on apoptosis in human neutrophils and lymphocytes. Special emphasis is given to assessment of membrane integrity of the cultured cells. Furthermore, a protocol for monitoring clearance of apoptotic neutrophils by monocyte-derived macrophages is provided.
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| 9. |
- Christenson, Karin, et al.
(author)
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Collection of in vivo transmigrated neutrophils from human skin.
- 2014
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In: Methods in molecular biology (Clifton, N.J.). - Totowa, NJ : Humana Press. - 1940-6029. ; 1124, s. 39-52
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Research review (peer-reviewed)abstract
- A wealth of knowledge on the life and death of human neutrophils has been obtained by the in vitro study of isolated cells derived from peripheral blood. However, neutrophils are of main importance, physiologically as well as pathologically, after they have left circulation and transmigrated to extravascular tissues. The journey from blood to tissue is complex and eventful, and tissue neutrophils are in many aspects distinct from the cells left in circulation. Here we describe how to obtain human tissue neutrophils in a controlled experimental setting from aseptic skin lesions created by the application of negative pressure. One protocol enables the direct analysis of the blister content, infiltrating leukocytes as well as exudate fluid, and is a simple method to follow multiple parameters of aseptic inflammation in vivo. Also described is the skin chamber technique, a method based on denuded skin blisters which are subsequently covered by collection chambers filled with autologous serum. Although slightly more artificial as compared to analysis of the blister content directly, the cellular yield of this skin chamber method is sufficient to perform a large number of functional analyses of in vivo transmigrated cells.
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| 10. |
- Dahlgren, Claes, 1949, et al.
(author)
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Measurement of respiratory burst products generated by professional phagocytes
- 2007
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In: Neutrophil Methods and Protocols. Mark T. QuinnFrank R. DeLeoGary M. Bokoch (red.). - Totowa, NJ : Springer. - 1064-3745 .- 1940-6029. - 9781588297884 ; , s. 349-63
-
Book chapter (peer-reviewed)abstract
- Activation of professional phagocytes, potent microbial killers of our innate immune system, is associated with an increase in cellular consumption of molecular oxygen (O2). The burst of 02 consumption is utilized by an NADPH-oxidase to generate highly-reactive oxygen species (ROS) starting with one and two electron reductions to generate superoxide anion (O2-) and hydrogen peroxide (H2O2), respectively. ROS are strongly bactericidal but may also cause tissue destruction and induce apoptosis in other immune competent cells of both the innate and the adaptive immune systems. Thus, the development of basic techniques to measure/quantify ROS generation/release by phagocytes during activation of the respiratory burst is of great importance, and a large number of techniques have been used for this purpose. Three of these techniques, chemiluminescence amplified by luminol/ isoluminol, the absorbance change following reduction of cytochrome c, and the fluorescence increase upon oxidation of p-hydroxyphenylacetate, are described in detail in this chapter. These techniques can be valuable tools in research spanning from basic phagocyte biology to more clinically-oriented research on innate immune mechanisms and inflammation.
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