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

Dette arbeidet ble gjort mulig ved NIH stipendet: R01GM109028 (S.P.C.)

1. Nilen rød (NR) flekker av lipider
<ol><li>Utarbeidelse av 5 mg/mL NR lagerløsning<ol>
<li>I en 500 mL flaske, legge 100 mg av NR pulver til 200 mL 100% aceton.</li>
		<li>Dekk flasken med aluminiumsfolie for å unngå noen eksponering for lys.</li>
		<li>Før bruk, rør løsningen for 2t i mørket.</li>
		<li>For langsiktig bruk, kan du lagre NR lager løsningen i en tett forseglet flaske uten noen lyseksponering. Skalere NR lagerløsning etter behov. Sikre at den samme lager løsningen brukes over NR flekker eksp...

<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. E., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemistry+and+the+worm:+Caenorhabditis+elegans+as+a+platform+for+integrating+chemical+and+biological+research.">Chemistry and the worm: Caenorhabditis elegans as a platform for integrating chemical and biological research.</a> Angew Chem Int Edit. 50 (21), 4774-4807 (2011).</li><li>Khanna, A., Johnson, D. L., 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=Physiological+roles+for+mafr-1+in+reproduction+and+lipid+homeostasis.">Physiological roles for mafr-1 in reproduction and lipid homeostasis.</a> Cell Rep. 9 (6), 2180-2191 (2014).</li><li>Lynn, D. A., Dalton, H. M., Sowa, J. N., Wang, M. C., Soukas, A. A., 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=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. E., Ruvkun, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+elegans+major+fats+are+stored+in+vesicles+distinct+from+lysosome-related+organelles.">C. elegans major fats are stored in vesicles distinct from lysosome-related organelles.</a> Cell Metab. 10 (5), 430-435 (2009).</li></ol>

Økningen i overvekt og metabolsk sykdom priser gjør C. elegans en passende modell å studere mekanismer som regulerer fett ansamling i celler og vev. Nyere bevis antyder at endringene i lipid nivåer er korrelert med cellulære prosesser fra insulin signalering8, aktivering av hormon reseptorer2, reproduktive utgang5. Rød O sammenlignet etikett-fri mikroskopi og kromatografi metoder, Nilen rød og olje og er relativt billig fargestoffer b...

Lipider er avgjørende for liv. De er integrert komponenter i membraner, fungere som sekundær budbringere og signalisere transduktorer, og har viktige funksjoner i energilagring. Når lipid metabolisme er dysregulated, fører det til sykdommer som fedme og type II diabetes, som trykker offentlig helse bekymringer9. Caenorhabditis elegans (C. elegans) er en utmerket modell organisme i å studere lipid metabolisme fordi den har en relativt kort livssyklus, en transparent kropp, en kjent celle avstamning og et fullt sekvensert genom. Først og fremst en Hermafroditt, C. elegans tillater forskere å heve store mengder isogenic dyr i korte perioder carryout høy gjennomstrømming frem genetisk skjermene å studere en rekke metabolske gener og veier4. Denne tilnærmingen har avsløre en høy grad av bevaring i 273 C. elegans lipid metabolisme gener blant mennesker, mus, rotter og drosophila. Videre har over 300 lipid gener i C. elegans menneskelige orthologues som er knyttet til sykdommer relatert til Metabolsk syndrom11. Tradisjonelt er undersøkelse av lipid lagring i C. elegans hovedsakelig basert på fargestoff-merket analyser, som gir robust opplysninger lipid akkumulering. En beskrivelse av hvor lipider lokalisere og målt forskjeller i lipid overflod er over vev. Men har nyere arbeid avdekket at lipid distribusjon kan være like viktig som lipid akkumulering6.
I det siste, studier har begynt å integrere metoder som høy ytelse flytende kromatografi-massespektrometri (såkalt HPLC-MS), gass kromatografi-massespektrometri (GC-MS), og sammenhengende anti-stokes Raman spredning (biler) mikroskopi adresse i mangler av flekken tilnærminger av direkte analysere innholdet av lipid ekstrakter, bestemte lipid fraksjoner og lipid innskudd, henholdsvis10,11. Videre har biler mikroskopi avdekket at Nilen røde kan bare tjene som en proxy for fett ansamling når den brukes som en etappe, den stabiliserende fargestoff, for sin bruk som en viktig flekken fører til off-målet farging av auto-fluorescerende organeller10. Men de nødvendige tekniske kompetanse og kostnadene forbundet med disse kromatografi og mikroskopi metoder gjør bruken uholdbar for mange forskning spørsmål. I denne artikkelen diskutere vi en praktisk og pålitelig metode for å fixate og flekken nøytral lipid innskudd i C. elegans bruker Nilen rød og olje røde O for å skille lipid overflod i hele dyr og bestemt vev.
Nilen rød, 9-diethylamino-5H-benzo[α]phenoxazine-5-one, er en benzophenoxazone farge som lett oppløses i ulike organiske løsemidler, men er mest uløselig i vann. Det er en utmerket lysochrome fargestoff brukt stain nøytral lipider som triglyserider eller kolesterol estere fordi den har en sterk farge, solubilizes i lipider, har ubetydelig samhandling med omkringliggende vev, og mindre løselig i løsemiddelet enn i lipider. Den har en eksitasjon og utslipp maxima 450-500 og 520 nm, henholdsvis1. Nilen rød-farget C. elegans er sett til grønne fluorescens, kan diskret lipid organer observeres under tarmen og andre vev enten i grupper eller jevnt spredt, avhengig av dyrets genotype eller eksperimentell behandling 7.
Olje røde O er en lysochrome, fettløselige fargestoff brukes stain triglyserider og lipoproteiner. Det kalles en azo fargestoff fordi den kjemiske strukturen inneholder to azo grupper knyttet til tre aromatiske ringer. Det er vanskelig å ionisere, noe som gjør det svært løselig i lipider. Dens lys absorpsjon maksimal er 518 nm 3flekken fargen er rød. C. elegans farget med olje røde O Vis røde lipid dråper som skiller seg ut mot dyr transparent kropp, som muliggjør kvalitativ vurdering av lipid fordeling mellom forskjellige vev6.

<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 er en utmerket modell organisme i å studere lipid metabolisme og energi homeostase. Mange av sine lipid gener er bevart i mennesker og er tilknyttet Metabolsk syndrom eller andre sykdommer. Undersøkelse av lipid akkumulering i denne organismen kan utføres av etappe, den stabiliserende fargestoffer eller etikett-fri metoder. Etappe, den stabiliserende flekker som Nile rød og olje røde O er rimelige, pålitelige måter å måle kvantitativt lipid nivåer og kvalitativt observere lipid distribusjon over vev, henholdsvis. Videre gir disse flekker høy gjennomstrømming screening av ulike lipid metabolisme gener og veier. I tillegg sine hydrofobe natur forenkler lipid løselighet, reduserer interaksjon med omkringliggende vev, og hindrer dissosiasjon i løsemiddelet. Disse metodene er effektiv på å undersøke de generelle lipid innhold, gir de ikke detaljert informasjon om kjemisk sammensetning og mangfoldet av lipid innskudd. For disse formål, etikett-fri metoder som GC-MS og biler mikroskopi er bedre egnet, kostnadene til tross.

SKN-1 er en bZip, cytoprotective transkripsjon faktor som aksjer homologi med pattedyr NRF2 og har vist seg å megle fettsyrer oksidering. Avhengig av glukose konsentrasjonen i kostholdet viser ormer med en constitutively aktivert skn-1 allelet forskjellige lipid nivåer når beiset med Nile røde7. Figur 1A C viser aktivert skn-1 dyr utsatt for forhold som fører til økende lipid nivåer. NR fluores...

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

Kvantifisering av Lipid overflod og evaluering av Lipid distribusjon i Caenorhabditis elegans av Nilen rød og olje Red O flekker

Nilen røde flekker av fast Caenorhabditis elegans er en metode for kvantitativ måling av nøytrale lipid innskudd, mens olje røde O flekker forenkler kvalitativ vurdering av lipid fordeling mellom vev.

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.

Research

JoVE Journal

Biology

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

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Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits ...

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A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This ...

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Lipid droplets (LDs) are organelles that play an important role in lipid metabolism and neutral lipid storage in cells. They are associated with a variety ...