Name: quantification of lipid abundance and evaluation of lipid distribution in caenorhabditis elegans by nile red and oil red o staining
Uploaded: 2018-03-05T14:00:00
Description: Watch this Scientific Journal Video about Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining at JoVE.com

This work was made possible by the NIH grant: R01GM109028 (S.P.C.)

1. Nile Red (NR) Staining of Lipids
<ol>
	<li>Preparation of 5 mg/mL NR stock solution
	<ol>
		<li>In a 500 mL bottle, add 100 mg of NR powder to 200 mL of 100% acetone.</li>
		<li>Cover the bottle with aluminum foil to avoid any exposure to light.</li>
		<li>Before use, stir the solution for 2 h in the dark.</li>
		<li>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 acr...

<ol>
	<li>Greenspan, P., Mayer, E. P., Fowler, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nile+red:+a+selective+fluorescent+stain+for+intracellular+lipid+droplets.">Nile red: a selective fluorescent stain for intracellular lipid droplets.</a> J Cell Biol. 100 (3), 965-973 (1985).</li><li>Guo, Y., Cordes, K. R., Farese, R. V., Walther, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+droplets+at+a+glance.">Lipid droplets at a glance.</a> J Cell Sci. 122 (6), 749-752 (2009).</li><li>Horobin, R. W., Kiernan, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Conn's biological stains: a handbook of dyes, stains and fluorochromes for use in biology and medicine. , (2002).</li><li>Hulme, S. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Omega-3+and-6+fatty+acids+allocate+somatic+and+germline+lipids+to+ensure+fitness+during+nutrient+and+oxidative+stress+in+Caenorhabditis+elegans.">Omega-3 and-6 fatty acids allocate somatic and germline lipids to ensure fitness during nutrient and oxidative stress in Caenorhabditis elegans.</a> P Natl Acad Sci USA. 112 (50), 15378-15383 (2015).</li><li>Pang, S., Lynn, D. A., Lo, J. Y., Paek, J., Curran, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SKN-1+and+Nrf2+couples+proline+catabolism+with+lipid+metabolism+during+nutrient+deprivation.">SKN-1 and Nrf2 couples proline catabolism with lipid metabolism during nutrient deprivation.</a> Nat Commun. 5, (2014).</li><li>Saltiel, A. R., Kahn, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+signalling+and+the+regulation+of+glucose+and+lipid+metabolism.">Insulin signalling and the regulation of glucose and lipid metabolism.</a> Nature. 414 (6865), 799-806 (2001).</li><li>Witting, M., Schmitt-Kopplin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Caenorhabditis+elegans+lipidome:+A+primer+for+lipid+analysis+in+Caenorhabditis+elegans.">The Caenorhabditis elegans lipidome: A primer for lipid analysis in Caenorhabditis elegans.</a> Arch Biochem Biophys. 589, 27-37 (2016).</li><li>Yen, K., Le, T. T., Bansal, A., Narasimhan, S. D., Cheng, J. X., Tissenbaum, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+of+fat+storage+quantitation+in+nematode+Caenorhabditis+elegans+using+label+and+label-free+methods.">A comparative study of fat storage quantitation in nematode Caenorhabditis elegans using label and label-free methods.</a> PloS One. 5 (9), e12810 (2010).</li><li>Zhang, Y., Zou, X., Ding, Y., Wang, H., Wu, X., Liang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+genomics+and+functional+study+of+lipid+metabolic+genes+in+Caenorhabditis+elegans.">Comparative genomics and functional study of lipid metabolic genes in Caenorhabditis elegans.</a> BMC Genomics. 14 (1), 164 (2013).</li><li>Folick, A., Min, W., Wang, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+imaging+of+lipid+dynamics+using+Coherent+Anti-stokes+Raman+Scattering+(CARS)+and+Stimulated+Raman+Scattering+(SRS)+microscopy.">Label-free imaging of lipid dynamics using Coherent Anti-stokes Raman Scattering (CARS) and Stimulated Raman Scattering (SRS) microscopy.</a> Curr Opin Genet Dev. 21 (5), 585-590 (2011).</li><li>Pino, E. C., Webster, C. M., Carr, C. E., Soukas, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+high+throughput+microscopic+assessment+of+fat+mass+in+Caenorhabditis+elegans.">Biochemical and high throughput microscopic assessment of fat mass in Caenorhabditis elegans.</a> J Vis Exp. (73), (2013).</li><li>O'Rourke, E. J., Soukas, A. A., Carr, C. 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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...

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 animals in short periods of time to carryout high-throughput forward genetic screens to study a wide array of metabolic genes and pathways4. This approach has revealed a high degree of conservation in 273 C. elegans lipid metabolism genes among humans, mice, rats and drosophila. Furthermore, over 300 lipid genes in C. elegans have human orthologues that are associated with diseases unrelated to metabolic syndrome11. Traditionally, examination of lipid storage in C. elegans has mostly relied on dye-labeled assays, which provide robust information about lipid accumulation. Less common is a description of where lipids localize and measured differences in lipid abundance across tissues. However, recent work has revealed that lipid distribution can be as important as lipid accumulation6.
Lately, studies have begun integrating methods such as high performance liquid chromatography-mass spectrometry (HPLC-MS), gas chromatography-mass spectrometry (GC-MS), and coherent anti-stokes Raman scattering (CARS) microscopy to address the shortcomings of stain-based approaches by directly analyzing the contents of lipid extracts, specific lipid fractions, and lipid deposits, respectively10,11. Moreover, CARS microscopy has revealed that Nile red can only serve as a proxy for fat accumulation when used as a fixative dye, for its use as a vital stain leads to off-target staining of auto-fluorescent organelles10. However, the required technical expertise and costs associated with these chromatography and microscopy methods make their use untenable for many research questions. In this article, we discuss a convenient and reliable method to fixate and stain neutral lipid deposits in C. elegans using Nile red and oil red O to distinguish lipid abundance in whole animals and in specific tissues.
Nile red, 9-diethylamino-5H-benzo[&#945;]phenoxazine-5-one, is a benzophenoxazone dye that readily dissolves in various organic solvents, but is mostly insoluble in water. It is an excellent lysochrome dye used to stain neutral lipids such as triglycerides or cholesterol esters because it features a strong color, solubilizes well in lipids, has negligible interaction with surrounding tissues, and is less soluble in the solvent than in lipids. It has an excitation and emission maxima of 450-500 and 520 nm, respectively1. When Nile red-stained C. elegans is viewed for green fluorescence, discrete lipid bodies can be observed throughout the intestine and other tissues either in clusters or evenly dispersed, depending on the animal&#39;s genotype or experimental treatment7.
Oil red O is a lysochrome, fat-soluble dye used to stain triglycerides and lipoproteins. It is called an azo dye because its chemical structure contains two azo groups attached to three aromatic rings. It is difficult to ionize, which renders it highly soluble in lipids. Its stain color is red and its light absorption maximum is 518 nm 3. C. elegans stained with oil red O show red lipid droplets that stand out against the animal&#39;s transparent body, which facilitates qualitative assessment of lipid distribution among different tissues6.

<table>
	<tbody>
		<tr>
			<td>Imager.M2m Microscope</td>
			<td>Zeiss</td>
			<td>n/a</td>
			<td>Fluorescence microscope</td>
		</tr>
		<tr>
			<td>ERC5s camera</td>
			<td>Axiocam</td>
			<td>n/a</td>
			<td>Color-capable</td>
		</tr>
		<tr>
			<td>MRm camera</td>
			<td>Axiocam</td>
			<td>n/a</td>
			<td>Fluorescence-capable</td>
		</tr>
		<tr>
			<td>Nile red</td>
			<td>Thermo Fisher</td>
			<td>N1142</td>
			<td>Lipid Stain</td>
		</tr>
		<tr>
			<td>Oil red O</td>
			<td>Alfa Aesar</td>
			<td>A12989</td>
			<td>Lipid Stain</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermo Fisher</td>
			<td>D1306</td>
			<td>DNA stain</td>
		</tr>
		<tr>
			<td>Isopropyl Alcohol</td>
			<td>BDH</td>
			<td>BDH1133-1LP</td>
			<td>Fixative solution</td>
		</tr>
		<tr>
			<td>0.2 &#181;m seterile syringe filter</td>
			<td>VWR</td>
			<td>28145-477</td>
			<td>Cellulose acetate filter</td>
		</tr>
		<tr>
			<td>Centrifuge 5430</td>
			<td>Eppendorf</td>
			<td>5428000015</td>
			<td>Centrifuge</td>
		</tr>
		<tr>
			<td>Shaker Rotisserie</td>
			<td>Lab Quake</td>
			<td>400110Q</td>
			<td>Shaker</td>
		</tr>
		<tr>
			<td>Tube Rotator</td>
			<td>VWR</td>
			<td>10136-084</td>
			<td>Rotator</td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Sigma-Aldrich</td>
			<td>7758-11-4</td>
			<td>NGM</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>7778-77-0</td>
			<td>NGM</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Alfa Aesar</td>
			<td>7786-30-3</td>
			<td>NGM</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>10035-04-8</td>
			<td>NGM</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>7647-14-5</td>
			<td>NGM</td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Sigma-Aldrich</td>
			<td>57-88-5</td>
			<td>NGM</td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>BD Biosciences</td>
			<td>211677</td>
			<td>NGM</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Teknova</td>
			<td>L9110</td>
			<td>NGM</td>
		</tr>
		<tr>
			<td>LB media</td>
			<td>Sigma-Aldrich</td>
			<td>L3147</td>
			<td>Bacterial growth</td>
		</tr>
	</tbody>
</table>

quantification of lipid abundance and evaluation of lipid distribution in caenorhabditis elegans by nile red and oil red o staining

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.

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...

Watch this Scientific Journal Video about Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining at JoVE.com

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

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.

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

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved ...

The overall goal of these staining methods is to quantify intracellular lipid abundance and evaluate lipid distribution among different tissues. This method can help answer key questions in lipid metabolism such as how are lipids genetically and biochemically regulated and which disease states this regulate lipid homeostasis. The main advantage of this technique is that it is relatively simple and inexpensive to carry out.To carry out Nile Red lipid staining, plate worms on Nematode Growth Medium or NGM seeded with late log OP50 E.coli and grow the worms at 20 degrees Celsius to early L4 stage. Wash the worms with one millimeter of PBST solution and transfer the worm suspension to a 1.5 milliliter microfuge tube. Centrifuge the worms at 560 times gravity for one minute.Then remove the supernatant and repeat the PBST wash until the E.coli is cleared from the suspension. Next, to the worm pellet, add 100 microliters of 40%isopropanol or 60%for ORO staining and incubate the sample at room temperature for three minutes. The addition of isopropanol is crucial for proper permeabilization of the worms before staining.Centrifuge the worms at 560 times gravity for one minute and remove the supernatant without disrupting the worm pellet. It is important to minimize light exposure during the centrifugation steps. Now while in the dark, add 600 microliters of previously prepared NR working solution to each sample.Invert the tubes three times and fully mix the worms and solution. Then incubate the sample in the dark at room temperature for two hours. Following incubation, centrifuge the worms and remove the supernatant.Then add 600 microliters of PBST and incubate the samples in the dark for 30 minutes to remove the excess NR stain. After spinning the samples again as before, remove all but approximately 50 microliters of supernatant and resuspend the worm pellet in the remaining solution. To prepare slides for imaging, pipette five microliters of worm suspension onto a microscope slide and carefully apply a coverslip to avoid trapping any air bubbles.Using the FITC GFP channel to image NR stained worms and trying different exposure times, image the worms at five-fold magnification to capture several animals per field of view. Then switch to 10-fold magnification for better quantification of individual worms. To quantify the images, upload the micrographs to ImageJ.In the image pull down menu, adjust the brightness, contrast of the image deck, and propagate changes to all the images by clicking apply. Use the polygon selection tool to delineate each imaged worm and under the analysis pull down menu, use the measure function to qualify fluorescence intensity emitted by NR.For each image, measure five background locations and calculate the average background fluorescence intensity. Add 600 microliters of Oil Red O or ORO working solution to each sample and invert the tube three times to mix the worms well and the ORO.Rotate the samples at 30 RPM and RT for two hours. Centrifuge the samples at 560 times gravity for one minute and aspirate all the supernatant except for 100 microliters. Then use 600 microliters of PBST to resuspend the samples and rotate the tubes at 30 RPM for 30 minutes to remove excess ORO staining.To better distinguish the location of the animals'germ line in the intestinal cells, stain nuclei by adding one microliter of DAPI for every one milliliter of ORO working solution. Then to prepare slides for imaging, resuspend the worms and the remaining supernatant and mix the solution well. Put five microliters of worm suspension on a microscope slide and carefully apply a coverslip ensuring that no air bubbles form.To image the ORO stained worms, use a color capable camera at five-fold magnification to image several worms in one field of view. Then switch to 10-fold magnification for better examination of individual worms. Finally, after uploading the images to ImageJ and creating an image deck according to the text protocol, categorize the images according to lipid accumulation throughout the animal's body or in specific tissues.SKN-1 shares homology with mammalian Nrf2 and has been shown to mediate fatty acid oxidation. This figure shows activated SKN-1 animals exposed to conditions that lead to increasing lipid levels. As seen in these images, NR fluorescence captured using the FITC GFP channel is prominent along the intestine, but is dimmer in the head, tail, and intestinal lumen.While it is easy to categorize the worms according to the presence or absence of Age-dependent Somatic Depletion of Fat or ASDF, it may be difficult to identify animals with intermediate fat loss. Compared to non-ASDF animals, which show bright red staining throughout the body with few translucent areas, ASDF worms exhibit noticeable fat depletion in intestinal cells. Animals in the process of developing ASDF often show translucent spots where most of the fat loss eventually occur.Moreover, ASDF worms may still feature bright red staining in the head and tail regions because the phenotype is not defined by complete somatic fat loss, but by extensive fat depletion relative to non-aged animals. Once mastered, this technique can be done in three hours plus imaging time if it is performed properly. While attempting this procedure, it's important to remember to wash and permeabilize worms no more than 30 minutes before staining.Following this procedure, other methods like CARS microscopy can be performed to compare the type of lipid species each technology identifies and to determine which method is better suited for specific types of questions. After watching this video, you should have a good understanding of how to quantify lipids and evaluate lipid distribution in the worms using Nile Red and Oil Red O staining, followed by ImageJ micrograph analysis.

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