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  • 1.
    Bergström Lind, Sara
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics.
    Artemenko, Konstantin A.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Pettersson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics.
    A strategy for identification of protein tyrosine phosphorylation2012In: Methods, ISSN 1046-2023, E-ISSN 1095-9130, Vol. 56, no 2, p. 275-283Article in journal (Refereed)
    Abstract [en]

    To develop methods for studying phosphorylation of protein tyrosine residues is an important task since this protein modification regulates many cellular functions and often is involved in oncogenesis. An optimal protocol includes enrichment of tyrosine phosphorylated (pTyr) peptides or proteins, followed by a high resolving analytical method for identification of the enriched components. In this Methods paper, we describe a working strategy on how immunoaffinity enrichments, using anti-pTyr antibodies, combined with mass spectrometric (MS) analysis can be used to study the pTyr proteome. We describe in detail how our procedure was used to characterize the pTyr proteome of K562 leukemia cells. Important questions concerning the use of different anti-pTyr antibodies, enrichments performed at the peptide and/or the protein level, pooling of enrichments and requirements for the MS characterization are discussed.

  • 2.
    Hauptmann, Giselbert
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Lauter, Gilbert
    Söll, Iris
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Detection and signal amplification in zebrafish RNA FISH2016In: Methods, ISSN 1046-2023, E-ISSN 1095-9130, Vol. 98, p. 50-59Article in journal (Refereed)
    Abstract [en]

    In situ hybridization (ISH) has become an invaluable tool for the detection of RNA in cells, tissues and organisms. Due to improvements in target and signal amplification and in probe design remarkable progress has been made concerning sensitivity, specificity and resolution of chromogenic and fluorescent ISH (FISH). These advancements allow for exquisite cellular and sub-cellular resolution and for detecting multiple RNA species at a time by multiplexing. In zebrafish (F)ISH non-enzymatic and enzymatic amplification systems have been employed to obtain enhanced signal intensities and signal-to-noise ratios. These amplification strategies include branched DNA-based RNAscope and in situ hybridization chain reaction (HCR) techniques, as well as alkaline phosphatase (AP)- and horseradish peroxidase (PO)-based immunoassays. For practical application, we provide proven multiplex FISH protocols for AP- and PO-based visualization of mRNAs at high resolution. The protocols take advantage of optimized tyramide signal amplification (TSA) conditions of the PO assay and long-lasting high signal-to-noise ratio of the AP reaction, thereby enabling detection of less abundant transcripts.

  • 3.
    Hober, Sophia
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Lindbo, Sarah
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Nilvebrant, Johan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Bispecific applications of non-immunoglobulin scaffold binders2019In: Methods, ISSN 1046-2023, E-ISSN 1095-9130, Vol. 154, p. 143-152Article in journal (Refereed)
    Abstract [en]

    Non-immunoglobulin scaffolds represent a proven group of small affinity proteins that can be engineered in vitro to similar affinity and potency as monoclonal antibodies. Several novel candidate biotherapeutics that exploit the potential advantages scaffold proteins hold over larger and more complex antibodies have been developed over the past decade. The ease of using small and robust binding proteins as flexible and modular building blocks has led to the development of a wide range of innovative approaches to combine them in various bi- and multispecific formats. This progress is expected to aid the ongoing challenge of identifying niche applications where clear differentiation from antibody-based molecules will be key to success. Given the many engineering options that are available for non-immunoglobulin scaffold proteins, they have potential to not only complement but probably also surpass antibodies in certain applications.

  • 4. Howat, William J
    et al.
    Lewis, Arthur
    Jones, Phillipa
    Kampf, Caroline
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Pontén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    van der Loos, Chris M
    Gray, Neil
    Womack, Chris
    Warford, Anthony
    Antibody validation of immunohistochemistry for biomarker discovery: Recommendations of a consortium of academic and pharmaceutical based histopathology researchers2014In: Methods, ISSN 1046-2023, E-ISSN 1095-9130, Vol. 70, no 1, p. 34-38Article in journal (Refereed)
    Abstract [en]

    As biomarker discovery takes centre-stage, the role of immunohistochemistry within that process is increasing. At the same time, the number of antibodies being produced for "research use" continues to rise and it is important that antibodies to be used as biomarkers are validated for specificity and sensitivity before use. This guideline seeks to provide a stepwise approach for the validation of an antibody for immunohistochemical assays, reflecting the views of a consortium of academic and pharmaceutical based histopathology researchers. We propose that antibodies are placed into a tier system, level 1-3, based on evidence of their usage in immunohistochemistry, and that the degree of validation required is proportionate to their place on that tier.

  • 5.
    Kanno, Takahiro
    et al.
    Department of Physiology, Hirosaki University School of Medicine, Hirosaki, Japan.
    Ma, Xiasong
    Department of Physiological Sciences, Lund University, Lund, Sweden.
    Barg, Sebastian
    Department of Physiological Sciences, Lund University, Lund, Sweden.
    Eliasson, Lena
    Department of Physiological Sciences, Lund University, Lund, Sweden.
    Galvanovskis, Juris
    Department of Physiological Sciences, Lund University, Lund, Sweden.
    Göpel, Sven
    Department of Physiological Sciences, Lund University, Lund, Sweden.
    Larsson, Max
    Department of Physiological Sciences, Lund University, Lund, Sweden.
    Renström, Erik
    Department of Physiological Sciences, Lund University, Lund, Sweden.
    Rorsman, Patrik
    Department of Physiological Sciences, Lund University, Lund, Sweden.
    Large dense-core vesicle exocytosis in pancreatic beta-cells monitored by capacitance measurements.2004In: Methods, ISSN 1046-2023, E-ISSN 1095-9130, Vol. 33, no 4, p. 302-311Article in journal (Refereed)
    Abstract [en]

    This article discusses the currently used methodologies for monitoring exocytosis as changes in cell capacitance. Details are given on composition of solutions, experimental protocols, and how the observed responses can be interpreted physiologically. The concepts are illustrated by examples from our own work on insulin-releasing pancreatic beta-cells. Finally, we consider the feasibility of applying capacitance measurements to endocrine cells in intact pancreatic islets, where the cells are electrically coupled to each other.

  • 6. Kanno, Takahiro
    et al.
    Ma, Xiasong
    Barg, Sebastian
    Eliasson, Lena
    Galvanovskis, Juris
    Göpel, Sven
    Larsson, Max
    Renström, Erik
    Rorsman, Patrik
    Large dense-core vesicle exocytosis in pancreatic beta-cells monitored by capacitance measurements.2004In: Methods, ISSN 1046-2023, E-ISSN 1095-9130, Vol. 33, no 4, p. 302-11Article in journal (Refereed)
    Abstract [en]

    This article discusses the currently used methodologies for monitoring exocytosis as changes in cell capacitance. Details are given on composition of solutions, experimental protocols, and how the observed responses can be interpreted physiologically. The concepts are illustrated by examples from our own work on insulin-releasing pancreatic beta-cells. Finally, we consider the feasibility of applying capacitance measurements to endocrine cells in intact pancreatic islets, where the cells are electrically coupled to each other.

  • 7.
    Lamichhane, Santosh
    et al.
    Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland.
    Sen, Partho
    Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland.
    Dickens, Alex M.
    Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland.
    Oresic, Matej
    Örebro University, School of Medical Sciences. Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland.
    Bertram, Hanne Christine
    Department of Food Science, Aarhus University, Aarslev, Denmark.
    Gut metabolome meets microbiome: A methodological perspective to understand the relationship between host and microbe2018In: Methods, ISSN 1046-2023, E-ISSN 1095-9130, Vol. 149, p. 3-12Article in journal (Refereed)
    Abstract [en]

    It is well established that gut microbes and their metabolic products regulate host metabolism. The interactions between the host and its gut microbiota are highly dynamic and complex. In this review we present and discuss the metabolomic strategies to study the gut microbial ecosystem. We highlight the metabolic profiling approaches to study faecal samples aimed at deciphering the metabolic product derived from gut microbiota. We also discuss how metabolomics data can be integrated with metagenomics data derived from gut microbiota and how such approaches may lead to better understanding of the microbial functions. Finally, the emerging approaches of genome-scale metabolic modelling to study microbial co-metabolism and host-microbe interactions are highlighted.

  • 8.
    Lind, Christoffer
    et al.
    Univ Maryland, Sch Pharm, Dept Pharmaceut Sci, Baltimore, MD 21201 USA.
    Esguerra, Mauricio
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Jespers, Willem
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Satpati, Priyadarshi
    Indian Inst Technol Guwahati, Dept Biosci & Bioengn, Gauhati 781039, Assam, India.
    Gutiérrez-de-Terán, Hugo
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Free energy calculations of RNA interactions2019In: Methods, ISSN 1046-2023, E-ISSN 1095-9130, Vol. 162-163, p. 85-95Article in journal (Refereed)
    Abstract [en]

    This review discusses the use of molecular dynamics free energy calculations for characterizing RNA interactions, with particular emphasis on molecular recognition events involved in mRNA translation on the ribosome. The general methodology for efficient free energy calculations is outlined and our specific implementation for binding free energy changes due to base mutations in mRNA and tRNA is described, We show that there are a number of key problems related to the accuracy of protein synthesis that can be addressed with this type of computational approach and several such examples are discussed in detail. These include the decoding of mRNA during peptide chain elongation, initiation and termination of translation, as well as the energetic effects of base tautomerization and tRNA modifications. It is shown that free energy calculations can be made sufficiently reliable to allow quantitative conclusions to be drawn regarding the energetics of cognate versus non-cognate interactions and its structural origins.

  • 9.
    Nilsson, Ola B.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Karolinska Institute, Sweden .
    van Hage, Marianne
    Grönlund, Hans
    Mammalian-derived respiratory allergens - Implications for diagnosis and therapy of individuals allergic to furry animals2014In: Methods, ISSN 1046-2023, E-ISSN 1095-9130, Vol. 66, no 1, p. 86-95Article in journal (Refereed)
    Abstract [en]

    Furry animals cause respiratory allergies in a significant proportion of the population. A majority of all mammalian allergens are spread as airborne particles, and several have been detected in environments where furry animals are not normally kept. The repertoire of allergens from each source belongs to a restricted number of allergen families. Classification of allergen families is particularly important for the characterization of allergenicity and cross-reactivity of allergens. In fact, major mammalian allergens are taken from only three protein families, i.e. the secretoglobin, lipocalin and kallikrein families. In particular, the lipocalin superfamily harbours major allergens in all important mammalian allergen sources, and cross-reactivity between lipocalin allergens may explain cross-species sensitization between mammals. The identification of single allergen components is of importance to improve diagnosis and therapy of allergic patients using component-resolved diagnostics and allergen-specific immunotherapy (ASIT) respectively. Major disadvantages with crude allergen extracts for these applications emphasize the benefits of careful characterization of individual allergens. Furthermore, detailed knowledge of the characteristics of an allergen is crucial to formulate attenuated allergy vaccines, e.g. hypoallergens. The diverse repertoires of individual allergens from different mammalian species influence the diagnostic potential and clinical efficacy of ASIT to furry animals. As such, detailed knowledge of individual allergens is essential for adequate clinical evaluation. This review compiles current knowledge of the allergen families of mammalian species, and discusses how this information may be used for improved diagnosis and therapy of individuals allergic to mammals.

  • 10.
    Persson, Camilla
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Kappert, Kai
    Engström, Ulla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Östman, Arne
    Sjöblom, Tobias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    An antibody-based method for monitoring in vivo oxidation of protein tyrosine phosphatases2005In: Methods, ISSN 1046-2023, E-ISSN 1095-9130, Vol. 35, no 1, p. 37-43Article in journal (Refereed)
    Abstract [en]

    Regulation of protein tyrosine phosphatases (PTPs) through reversible oxidation of the active site cysteine is emerging as a general, yet poorly characterized, mechanism for control of the activity of this important group of enzymes. This regulatory mechanism was initially described after in vitro treatment of PTPs with oxidizing agents. However, accumulating evidence has substantiated the notion that this mechanism is also operating in vivo, e.g., in association with the transient increase in H(2)O(2) production which occurs after activation of receptor tyrosine kinases. A novel generic antibody-based method for monitoring of PTP oxidation is described. The sensitivity of this strategy has been validated by the demonstration of oxidation of endogenously expressed PTPs after stimulation of cells with growth factors. The method was also instrumental in providing the first evidence for intrinsic differences between PTP domains with regard to sensitivity to oxidation.

  • 11.
    Shariatgorji, Mohammadreza
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Nilsson, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Bonta, Maximilian
    TU Wien, Inst Chem Technol & Analyt, Vienna, Austria..
    Gan, Jinrui
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Fudan Univ, Dept Chem, Inst Biomed Sci, Shanghai, Peoples R China.;Fudan Univ, State Key Lab Mol Engn Polymers, Shanghai, Peoples R China..
    Marklund, Niklas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Neurosurgery.
    Clausen, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Neurosurgery.
    Kallback, Patrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Lodén, Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Limbeck, Andreas
    TU Wien, Inst Chem Technol & Analyt, Vienna, Austria..
    Andrén, Per E.
    Direct imaging of elemental distributions in tissue sections by laser ablation mass spectrometry2016In: Methods, ISSN 1046-2023, E-ISSN 1095-9130, Vol. 104, p. 86-92Article in journal (Refereed)
    Abstract [en]

    We present a strategy for imaging of elements in biological tissues using laser ablation (LA) mass spectrometry (MS), which was compared to laser ablation inductively coupled plasma (LA-ICP) MS. Both methods were adopted for quantitative imaging of elements in mouse kidney, as well as traumatic brain injury model tissue sections. MS imaging (MSI) employing LA provides quantitative data by comparing signal abundances of sodium from tissues to those obtained by imaging quantitation calibration standards of the target element applied to adjacent control tissue sections. LA-ICP MSI provided quantitative data for several essential elements in both brain and kidney tissue sections using a dried-droplet approach. Both methods were used to image a rat model of traumatic brain injury, revealing accumulations of sodium and calcium in the impact area and its peripheral regions. LA MSI is shown to be a viable option for quantitative imaging of specific elements in biological tissue sections.

  • 12.
    Söderberg, Ola
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Leuchowius, Karl-Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Gullberg, Mats
    Jarvius, Malin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Weibrecht, Irene
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Larsson, Lars-Gunnar
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Characterizing proteins and their interactions in cells and tissues using the in situ proximity ligation assay.2008In: Methods, ISSN 1046-2023, E-ISSN 1095-9130, Vol. 45, no 3, p. 227-32Article in journal (Refereed)
    Abstract [en]

    The activity of proteins is typically regulated by secondary modifications and by interactions with other partners, resulting in the formation of protein complexes whose functions depend on the participating proteins. Accordingly, it is of central importance to monitor the presence of interaction complexes as well as their localization, thus providing information about the types of cells where the proteins are located and in what sub-cellular compartment these interactions occur. Several methods for visualizing protein interactions in situ have been developed during the last decade. These methods in most cases involve genetic constructs, and they have been successfully used in assays of living cell maintained in tissue culture, but they cannot easily be implemented in studies of clinical specimens. For such samples, affinity reagents like antibodies can be used to target the interacting proteins. In this review we will describe the in situ proximity ligation assays (in situ PLA), a method that is suitable for visualizing protein interactions in both tissue sections and in vitro cell lines, and we discuss research tasks when this or other method may be selected.

  • 13.
    Tornmalm, Johan
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.
    Label-free monitoring of ambient oxygenation and redox conditions using the photodynamics of flavin compounds and transient state (TRAST) spectroscopy2018In: Methods, ISSN 1046-2023, E-ISSN 1095-9130, Vol. 140, p. 178-187Article in journal (Refereed)
    Abstract [en]

    Transient state (TRAST) monitoring can determine population dynamics of long-lived, dark transient states of fluorescent molecules, detecting only the average fluorescence intensity from a sample, when subject to different excitation pulse trains. Like Fluorescence Correlation Spectroscopy (FCS), TRAST unites the detection sensitivity of fluorescence with the environmental sensitivity of long-lived non-fluorescent states, but does not rely on detection of stochastic fluorescence fluctuations from individual molecules. Relaxed requirements on noise suppression, detection quantum yield and time-resolution of the instrument, as well as on fluorescence brightness of the molecules studied, make TRAST broadly applicable, opening also for investigations based on less bright, auto-fluorescent molecules. In this work, we applied TRAST to study the transient state population dynamics within the auto-fluorescent coenzymes flavin adenine dinucleotide (FAD) and flavin-mononucleotide (FMN). From the experimental TRAST data, we defined state models, and determined rate parameters for triplet state and redox transitions within FMN and FAD, stacking and un-stacking rates of external redox active quenching agents and by the adenine moiety of FAD itself. TRAST experiments were found to be well capable to resolve these transitions in FMN and FAD, and to track how the transitions are influenced by ambient oxygenation and redox conditions. This work demonstrates that TRAST provides a useful tool to follow local oxygenation and redox conditions via FMN and FAD fluorescence, and forms the basis for measurements on flavoproteins and of redox and metabolic conditions in more complex environments, such as in live cells.

  • 14.
    Volkov, Ivan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Seefeldt, A. Carolin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Johansson, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Tracking of single tRNAs for translation kinetics measurements in chloramphenicol treated bacteria2019In: Methods, ISSN 1046-2023, E-ISSN 1095-9130, Vol. 162-163, p. 23-30Article in journal (Refereed)
    Abstract [en]

    Chloramphenicol is a broad-spectrum antibiotic targeting the protein synthesis machinery by binding to the bacterial ribosome. Chloramphenicol has been considered a classic general inhibitor of translation, blocking the accommodation of aa-tRNA into the A site of the large ribosomal subunit. However, recent studies suggest that this proposed mechanism is a simplification and that the effect of chloramphenicol on mRNA translation is much more dynamic. By tracking single dye-labelled elongator and initiator tRNAs in Escherichia coli cells treated with chloramphenicol, we observe the direct effect of chloramphenicol on translation kinetics. We find clear indications of slow but significant mRNA translation on drug bound ribosomes.

  • 15.
    Wennmalm, Stefan
    KTH, School of Engineering Sciences (SCI), Applied Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Potentials and pitfalls of inverse fluorescence correlation spectroscopy2018In: Methods, ISSN 1046-2023, E-ISSN 1095-9130, Vol. 140, p. 23-31Article in journal (Refereed)
    Abstract [en]

    Inverse Fluorescence Correlation Spectroscopy (iFCS) is a variant of FCS where unlabeled particles in solution, or domains in membranes, displace their surrounding, signal-generating molecules and thereby generate fluctuations. iFCS has to date been applied to unlabeled as well as labeled particles and protein molecules, using fluorescence as well as Raman scattering as a signal source, in diffraction-limited detection volumes as well as in nano-wells, and on fixed surfaces as well as in lipid bilayers. This review describes these applications and discusses the potentials and pitfalls when using iFCS.

  • 16.
    Wählby, Carolina
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Visual Information and Interaction. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis and Human-Computer Interaction. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Conery, Annie Lee
    Bray, Mark-Anthony
    Kamentsky, Lee
    Larkins-Ford, Jonah
    Sokolnicki, Katherine L.
    Veneskey, Matthew
    Michaels, Kerry
    Carpenter, Anne E.
    O'Rourke, Eyleen J.
    High- and low-throughput scoring of fat mass and body fat distribution in C. elegans2014In: Methods, ISSN 1046-2023, E-ISSN 1095-9130, Vol. 68, no 3, p. 492-499Article in journal (Refereed)
    Abstract [en]

    Fat accumulation is a complex phenotype affected by factors such as neuroendocrine signaling, feeding, activity, and reproductive output. Accordingly, the most informative screens for genes and compounds affecting fat accumulation would be those carried out in whole living animals. Caenorhabditis elegans is a well-established and effective model organism, especially for biological processes that involve organ systems and multicellular interactions, such as metabolism. Every cell in the transparent body of C. elegans is visible under a light microscope. Consequently, an accessible and reliable method to visualize worm lipid-droplet fat depots would make C. elegans the only metazoan in which genes affecting not only fat mass but also body fat distribution could be assessed at a genome-wide scale. Here we present a radical improvement in oil red O worm staining together with high-throughput image-based phenotyping. The three-step sample preparation method is robust, formaldehyde-free, and inexpensive, and requires only 15 min of hands-on time to process a 96-well plate. Together with our free and user-friendly automated image analysis package, this method enables C. elegans sample preparation and phenotype scoring at a scale that is compatible with genome-wide screens. Thus we present a feasible approach to small-scale phenotyping and large-scale screening for genetic and/or chemical perturbations that lead to alterations in fat quantity and distribution in whole animals.

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