Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Summary

Nile red staining of fixed Caenorhabditis elegans is a method for quantitative measurement of neutral lipid deposits, while oil red O staining facilitates qualitative assessment of lipid distribution among tissues.

Abstract

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved in humans and are associated with metabolic syndrome or other diseases. Examination of lipid accumulation in this organism can be carried out by fixative dyes or label-free methods. Fixative stains like Nile red and oil red O are inexpensive, reliable ways to quantitatively measure lipid levels and to qualitatively observe lipid distribution across tissues, respectively. Moreover, these stains allow for high-throughput screening of various lipid metabolism genes and pathways. Additionally, their hydrophobic nature facilitates lipid solubility, reduces interaction with surrounding tissues, and prevents dissociation into the solvent. Though these methods are effective at examining general lipid content, they do not provide detailed information about the chemical composition and diversity of lipid deposits. For these purposes, label-free methods such as GC-MS and CARS microscopy are better suited, their costs notwithstanding.

Introduction

Lipids are essential for life. They are integral components of membranes, act as secondary messengers and signal transducers, and have crucial functions in energy storage. When lipid metabolism is dysregulated, it leads to diseases like obesity and type II diabetes, which are pressing public health concerns⁹. Caenorhabditis elegans (C. elegans) is an excellent model organism in which to study lipid metabolism because it has a relatively short life cycle, a transparent body, a known cell lineage, and a fully sequenced genome. Primarily a hermaphrodite, C. elegans allows researchers to raise large numbers of isogenic an....

Protocol

1. Nile Red (NR) Staining of Lipids

1. Preparation of 5 mg/mL NR stock solution
   1. In a 500 mL bottle, add 100 mg of NR powder to 200 mL of 100% acetone.
   2. Cover the bottle with aluminum foil to avoid any exposure to light.
   3. Before use, stir the solution for 2 h in the dark.
   4. For long term use, store the NR stock solution in a tightly sealed bottle without any light exposure. Scale NR stock solution according to research needs. Ensure that the same stock solution is used across NR-staining experiments to obtain consistent staining and imaging results.
2. Preparation of NR working solution
   1. ....

Results

SKN-1 is a bZip, cytoprotective transcription factor that shares homology with mammalian NRF2 and has been shown to mediate fatty acid oxidation. Depending on the glucose concentration in their diet, worms with a constitutively activated skn-1 allele show different lipid levels when stained with Nile red⁷. Figure 1A-C shows activated skn-1 animals exposed to conditions that lead to increasing lipid le.......

Discussion

The rise in obesity and metabolic disease rates makes C. elegans a suitable model to study the mechanisms that regulate fat accumulation in cells and tissues. Recent evidence suggests that the changes in lipid levels are correlated with cellular processes ranging from insulin signaling⁸, the activation of hormone receptors², to reproductive output⁵. Compared to label-free microscopy and chromatography methods, Nile red and oil red O are rela.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Imager.M2m Microscope	Zeiss	n/a	Fluorescence microscope
ERC5s camera	Axiocam	n/a	Color-capable
MRm camera	Axiocam	n/a	Fluorescence-capable
Nile red	Thermo Fisher	N1142	Lipid Stain
Oil red O	Alfa Aesar	A12989	Lipid Stain
DAPI	Thermo Fisher	D1306	DNA stain
Isopropyl Alcohol	BDH	BDH1133-1LP	Fixative solution
0.2 µm seterile syringe filter	VWR	28145-477	Cellulose acetate filter
Centrifuge 5430	Eppendorf	5428000015	Centrifuge
Shaker Rotisserie	Lab Quake	400110Q	Shaker
Tube Rotator	VWR	10136-084	Rotator
K2HPO4	Sigma-Aldrich	7758-11-4	NGM
KH2PO4	Sigma-Aldrich	7778-77-0	NGM
MgSO4	Alfa Aesar	7786-30-3	NGM
CaCl2	Sigma-Aldrich	10035-04-8	NGM
NaCl	Sigma-Aldrich	7647-14-5	NGM
Cholesterol	Sigma-Aldrich	57-88-5	NGM
Peptone	BD Biosciences	211677	NGM
Agar	Teknova	L9110	NGM
LB media	Sigma-Aldrich	L3147	Bacterial growth