title: Fluorescence-based fixative and vital staining of lipid droplets in Caenorhabditis elegans reveal fat stores using microscopy and flow cytometry approaches.
abstract: The proportions of body fat and fat-free mass are determining factors of adiposity-associated diseases. Work in Caenorhabditis elegans has revealed evolutionarily conserved pathways of fat metabolism. Nevertheless, analysis of body composition and fat distribution in the nematodes has only been partially unraveled because of methodological difficulties. We characterized metabolic C. elegans mutants by using novel and feasible BODIPY 493/503-based fat staining and flow cytometry approaches. Fixative as well as vital BODIPY staining procedures visualize major fat stores, preserve native lipid droplet morphology, and allow quantification of fat content per body volume of individual worms. Colocalization studies using coherent anti-Stokes Raman scattering microscopy, Raman microspectroscopy, and imaging of lysosome-related organelles as well as biochemical measurement confirm our approaches. We found that the fat-to-volume ratio of dietary restriction, TGF-β, and germline mutants are specific for each strain. In contrast, the proportion of fat-free mass is constant between the mutants, although their volumes differ by a factor of 3. Our approaches enable sensitive, accurate, and high-throughput assessment of adiposity in large C. elegans populations at a single-worm level.
authors.affiliation: Institute of Human Nutrition and Food Science, Research Group Molecular Prevention, University of Kiel, Kiel, Germany.
authors.forename: Maja
authors.initials: M
authors.lastname: Klapper
authors.affiliation: Institute of Human Nutrition and Food Science, Research Group Molecular Prevention, University of Kiel, Kiel, Germany.
authors.forename: Madeleine
authors.initials: M
authors.lastname: Ehmke
authors.affiliation: Institute of Human Nutrition and Food Science, Research Group Molecular Prevention, University of Kiel, Kiel, Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Palgunow
authors.affiliation: Institute of Human Nutrition and Food Science, Research Group Molecular Prevention, University of Kiel, Kiel, Germany.
authors.forename: Mike
authors.initials: M
authors.lastname: Böhme
authors.affiliation: Institute of Photonic Technology, Jena, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Matthäus
authors.affiliation: Institute of Photonic Technology, Jena, Germany.
authors.forename: Gero
authors.initials: G
authors.lastname: Bergner
authors.affiliation: Institute of Photonic Technology, Jena, Germany.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Dietzek
authors.affiliation: Institute of Photonic Technology, Jena, Germany.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Popp
authors.affiliation: Institute of Human Nutrition and Food Science, Research Group Molecular Prevention, University of Kiel, Kiel, Germany. Electronic address: fdoering@molprev.uni-kiel.de.
authors.forename: Frank
authors.initials: F
authors.lastname: Döring
mesh_terms: D000273:Adipose Tissue / Q000737:chemistry / Q000378:metabolism*; D050154:Adiposity; D000818:Animals; D001391:Azo Compounds / Q000032:analysis; D001896:Boron Compounds / Q000032:analysis; D017173:Caenorhabditis elegans / Q000235:genetics / Q000378:metabolism*; D004195:Disease Models, Animal; D005404:Fixatives / Q000032:analysis / Q000378:metabolism; D005434:Flow Cytometry; D005453:Fluorescence; D018095:Germ-Line Mutation; D057166:High-Throughput Screening Assays; D050356:Lipid Metabolism; D008853:Microscopy; D009765:Obesity / Q000378:metabolism*; D013045:Species Specificity; D013059:Spectrum Analysis, Raman; D013194:Staining and Labeling / Q000379:methods*; D016212:Transforming Growth Factor beta / Q000032:analysis / Q000096:biosynthesis
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001391:Azo Compounds; D001896:Boron Compounds; D005404:Fixatives; D016212:Transforming Growth Factor beta; C011049:oil red O