Fluorescence-based fixative and vital staining of lipid droplets in Caenorhabditis elegans reveal fat stores using microscopy and flow cytometry approaches

Abstract

Body composition with respect to white adipose tissue and lean body mass is of central importance and a determining factor of adiposity-associated perturbations, such as diabetes type 2, cardiovascular disease, and cancer. Therefore, modulation of body composition and especially the reduction of body fat are the main foci of dietary and lifestyle interventions. In the last few years, Caenorhabditis elegans became a model organism for investigating fat storage and metabolism, as reviewed by different authors ( 1-3 ). Based on forward genetics ( 4-6 ), functional genomics, and candidate gene approaches ( 7,8 ), more than 400 genes possibly involved in fat storage were described in C. elegans . Most of these genes are evolutionarily conserved and allocated in common pathways and are of relevance for the study of human adiposity.Although C. elegans was extensively used as a model organism to study the genetic and functional basis of fat storage, robust lipid droplet markers such as perilipin are not available at the moment ( 2 ). Appropriate methods concerning the distribution behavior of dyes in the worm or the reproducibility of staining procedures are still under discussion ( 3 ). The classical fi xative Sudan black dye ( 9 ) is shown to be very error prone in C. elegans because of the last ethanol-based washing steps. Recently O' Rourke et al. ( 10 ) demonstrated that the vital dyes Nile red and C1-C12-BODIPY-labeled fatty acid, which were used in more than 75 reports, stain lysosome-like granules rather than lipid droplets within the C. elegans intestine. These dyes were shown not to localize with the fi xative neutral fat fl uorophore LipidTox ( 10 ) 17 March 2011. Published, JLR Papers in Press, March 18, 2011 DOI 10.1194 Fluorescence-based fi xative and vital staining of lipid droplets in Caenorhabditis elegans reveal fat stores using microscopy and fl ow cytometry approaches Abbreviations: BP, bandpass; CARS, coherent anti-Stokes Raman scattering; COPAS, cytometry-based object parametric analysis and sorting system; LRO, lysosome-related organelle; OPO, optical parametric oscillator; TOF, time of fl ight; TAG, triacylglycerol. Manuscript received 8 October 2010 and in revised form

Paternal dietary restriction affects progeny fat content in Caenorhabditis elegans

“…Next, we quantified fat content in the offspring of food-restricted males using a fixative BODIPY TM 493/503 staining procedure. The accuracy of our fat staining method was confirmed by colocalization studies using CARS and Raman microscopy, imaging of lysosome-related organelles, and biochemical measurement (25). We found an inverted U-shaped relationship between the extent of paternal dietary restriction and fat content of progeny (Fig.…”

“…Worms were kept on nematode growth medium (NGM) agar plates seeded with E. coli OP50 at 20 8C (24). For all experiments, adult worms were treated with hypochlorite and then a definite number of eggs were sorted on plates using a COPAS flow cytometry instrument equipped as described (25). The gain signals for extinction, green, yellow, and red were set at 1.…”

“…Worms were harvested and fixative stained with BODIPY TM 493/503 as described (25). BODIPY-stained worms were imaged with an Axio Observer D1 (Zeiss, Jena, Germany) inverted microscope using an attached Axiocam MRm camera (Zeiss).…”

S-Adenosyl methionine synthetase 1 limits fat storage in Caenorhabditis elegans

“…To visualize fat storage in eggs, synchronized L4 larvae, and adult worms, vital BODIPY TM 493/503 (Invitrogen, Darmstadt, Germany) staining was performed as previously described (Klapper et al 2011). Briefly, freshly harvested worms were washed 3 times with M9 buffer and incubated in 500 ll of BODIPY TM 493/503 solution (6.7 lg/ll M9 buffer) for 20 min.…”

“…To determine the number and size of lipid droplets, the BODIPY TM 493/503 fixative fat staining method was applied as previously described (Klapper et al 2011). Briefly, freshly harvested worms were washed with M9 and fixed in a 4 % paraformaldehyde solution for 15 min.…”