Name: analysis of neutral lipid synthesis in saccharomyces cerevisiae by metabolic labeling and thin layer chromatography
Uploaded: 2021-02-02T13:30:00
Description: Watch this Scientific Journal Video about Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography at JoVE.com

The authors would like to thank the members of the Henne lab for help and conceptual advice in the completion of this study. W.M.H. is supported by funds from the Welch Foundation (I-1873), the NIH NIGMS (GM119768), the Ara Paresghian Medical Research Fund, and the UT Southwestern Endowed Scholars Program. S.R has been supported by a T32 program grant (5T32GM008297).

1. Growth and labeling of yeast cells with 14C-acetic acid
<ol>
	<li>Inoculate a yeast culture by picking a colony from a plate and dispensing it into 20 mL of synthetic complete (SC) media containing 2% dextrose (see Supplementary File for the recipe of SC media). Incubate at 30 &#730;C for overnight with shaking at 200 rpm. 
	NOTE: Growth condition, sample volume, and treatment will differ based on the lipid(s) of interest. Prior to running full experiments, optimal growth conditions ...

<ol>
	<li>Konige, M., Wang, H., Sztalryd, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+adipose+specific+lipid+droplet+proteins+in+maintaining+whole+body+energy+homeostasis.">Role of adipose specific lipid droplet proteins in maintaining whole body energy homeostasis.</a> Biochimica Et Biophysica Acta. 1842 (3), 393-401 (2014).</li><li>Arrese, E. L., Saudale, F. Z., Soulages, J. L. <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+as+signaling+platforms+linking+metabolic+and+cellular+functions.">Lipid droplets as signaling platforms linking metabolic and cellular functions.</a> Lipid Insights. 7, 7-16 (2014).</li><li>Walther, T. C., Chung, J., Farese, R. 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K., Bates, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+phase+HPLC+method+for+combined+separation+of+both+polar+and+neutral+lipid+classes+with+application+to+lipid+metabolic+flux.">Normal phase HPLC method for combined separation of both polar and neutral lipid classes with application to lipid metabolic flux.</a> Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences. 1145, 122099 (2020).</li><li>Knittelfelder, O. L., Weberhofer, B. P., Eichmann, T. O., Kohlwein, S. D., Rechberger, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+versatile+ultra-high+performance+LC-MS+method+for+lipid+profiling.">A versatile ultra-high performance LC-MS method for lipid profiling.</a> Journal of Chromatography B. 951-952, 119-128 (2014).</li><li>Ruiz, J. I., Ochoa, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+in+the+subnanomolar+range+of+phospholipids+and+neutral+lipids+by+monodimensional+thin-layer+chromatography+and+image+analysis.">Quantification in the subnanomolar range of phospholipids and neutral lipids by monodimensional thin-layer chromatography and image analysis.</a> Journal of Lipid Research. 38 (7), 1482-1489 (1997).</li><li>Bui, Q., Sherma, J., Hines, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+high+performance+thin+layer+chromatography-densitometry+to+study+the+influence+of+the+prion+[RNQ+]+and+its+determinant+prion+protein+Rnq1+on+yeast+lipid+profiles.">Using high performance thin layer chromatography-densitometry to study the influence of the prion [RNQ+] and its determinant prion protein Rnq1 on yeast lipid profiles.</a> Separations. 5 (1), 5010006 (2018).</li><li>Pronk, J. T., de Steensma, H., Van Dijken, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyruvate+metabolism+in+Saccharomyces+cerevisiae.">Pyruvate metabolism in Saccharomyces cerevisiae.</a> Yeast. 12 (16), 1607-1633 (1996).</li><li>Buttke, T. M., Pyle, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+unsaturated+fatty+acid+deprivation+on+neutral+lipid+synthesis+in+Saccharomyces+cerevisiae.">Effects of unsaturated fatty acid deprivation on neutral lipid synthesis in Saccharomyces cerevisiae.</a> Journal of Bacteriology. 152 (2), 747-756 (1982).</li><li>Touchstone, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thin-layer+chromatographic+procedures+for+lipid+separation.">Thin-layer chromatographic procedures for lipid separation.</a> Journal of Chromatography B: Biomedical Sciences and Applications. 671 (1-2), 169-195 (1995).</li><li>Folch, J., Lees, M., Sloane Stanley, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+the+isolation+and+purification+of+total+lipides+from+animal+tissues.">A simple method for the isolation and purification of total lipides from animal tissues.</a> The Journal of Biological Chemistry. 226 (1), 497-509 (1957).</li><li>Breil, C., Abert Vian, M., Zemb, T., Kunz, W., Chemat, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Bligh+and+Dyer"+and+Folch+methods+for+solid-liquid-liquid+extraction+of+lipids+from+microorganisms.+Comprehension+of+solvatation+mechanisms+and+towards+substitution+with+alternative+solvents.">"Bligh and Dyer" and Folch methods for solid-liquid-liquid extraction of lipids from microorganisms. Comprehension of solvatation mechanisms and towards substitution with alternative solvents.</a> International Journal of Molecular Sciences. 18 (4), 708 (2017).</li><li>Fuchs, B., S&#252;&#223;, R., Teuber, K., Eibisch, M., Schiller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+analysis+by+thin-layer+chromatography-A+review+of+the+current+state.">Lipid analysis by thin-layer chromatography-A review of the current state.</a> Journal of Chromatography A. 1218 (19), 2754-2774 (2011).</li></ol>

Here, a versatile radiolabeling protocol to quantitatively monitor the synthesis of NL species in yeast is presented. This protocol is very modular, which allows for the procedure to be finished within 3-6 days. Additionally, a wealth of literature exists on the use of TLC to separate lipid species and metabolites, which should permit the user to detect several lipid species of interest with a simple change of TLC solvent systems16,19.This protocol is conducive t...

Acetyl-CoA is the fundamental building block of diverse biomolecules including neutral lipids (NLs), which serve as a versatile biomolecular currency for building membranes, generating ATP, and regulating cell signaling1,2. The availability of NLs to be shunted into any of these respective pathways is, in part, regulated by their storage. Lipid droplets (LDs), cytoplasmic organelles composed of hydrophobic cores of triglycerides (TGs) and sterol-esters (SEs), are the main storage compartments of most cellular NLs. As such, LDs sequester and regulate NLs, which can be degraded and subsequently utilized for biochemical and metabolic processes3,4. It is known that the mis-regulation of NL and LD-associated proteins is correlated with the onset of pathologies including lipodystrophy and metabolic syndromes5,6. Because of this, current LD research is intensely focused on how NL synthesis is regulated spatially, temporally, and across distinct tissues of multi-cellular organisms. Due to the ubiquitous cellular roles for NLs, many enzymes responsible for the synthesis and regulation of NLs are conserved throughout eukaryotes7. Indeed, even some prokaryotes store NLs in LDs8. Therefore, genetically tractable model organisms such as Saccharomyces cerevisiae (budding yeast) have been useful for the study of NL synthesis and regulation.
The separation and quantification of NLs from cell extracts can be accomplished in a myriad of ways, including gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC), and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS)9,10,11. Perhaps the simplest method for separating NLs is via thin layer chromatography (TLC), which allows for subsequent densitometric quantification from a standard curve12,13. Although TLC provides only a course-grained separation of NLs, it remains a powerful technique because it is inexpensive, and it allows for the rapid separation of NLs from several samples simultaneously. Two of the most considerable challenges facing the study of NLs via TLC are: 1) the broad range of cellular abundances of NL species and their intermediates, and 2) the range of hydrophilicity/hydrophobicity of lipid intermediates within NL synthesis pathways. Consequently, the quantification of NL species via TLC is typically restricted to the most abundant species; however, introduction of a 14C-acetic acid radiolabel can significantly enhance the detection of low abundance intermediates within NL pathways. Acetic acid is rapidly converted into acetyl-CoA by the acetyl-CoA synthetase ACS214, which makes 14C-acetic acid a suitable radiolabeling substrate in yeast15. Additionally, separation of both hydrophobic NLs and hydrophilic intermediates of NLs can be achieved by TLC through the use of multiple solvent systems16. Here, a method is presented for the separation of NLs using 14C-acetic acid metabolic labeling in yeast. Lipids labeled during the pulse period are subsequently isolated by a well-established total lipid isolation protocol17, followed by the separation of NL species by TLC. Developing of TLC plates by both autoradiography to visualize labeled lipids, and a chemical spray to visualize total lipids, permits for multiple methods of quantification. Individual lipid bands can also be easily extracted from the TLC plate using a razor blade, and scintillation counting can be used to quantify amount of radiolabeled material within the band.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>[1-C14] Acetic acid sodium salt specific activity: 45-60mCi</td>
			<td>PerkinElmer</td>
			<td>NEC084H001MC</td>
			<td></td>
		</tr>
		<tr>
			<td>18:1 1,2 dioleoyl-sn-glycerol</td>
			<td>Avanti</td>
			<td>800811O</td>
			<td></td>
		</tr>
		<tr>
			<td>200 proof absolute ethanol</td>
			<td>Sigma</td>
			<td>459836</td>
			<td></td>
		</tr>
		<tr>
			<td>Acid washed glass beads 425-600um</td>
			<td>Sigma</td>
			<td>G8772</td>
			<td></td>
		</tr>
		<tr>
			<td>Amber bulbs for Pastuer pipettes</td>
			<td>Fisher</td>
			<td>03-448-24</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Sulfate &#62;99%</td>
			<td>Sigma</td>
			<td>A4418</td>
			<td></td>
		</tr>
		<tr>
			<td>Beckman LS6500 scintillation counter</td>
			<td>PerkinElmer</td>
			<td>A481000</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform (HPLC grade)</td>
			<td>Fisher</td>
			<td>C607SK</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol &#62;99%</td>
			<td>Sigma</td>
			<td>C8667</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesteryl-linoleate &#62;98%</td>
			<td>Sigma</td>
			<td>C0289</td>
			<td></td>
		</tr>
		<tr>
			<td>Concentrated sulfuric acid</td>
			<td>Sigma</td>
			<td>339741</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 50mL conical tubes, polypropylene with centristar cap</td>
			<td>Sigma</td>
			<td>CLS430829</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose, anhydrous grade</td>
			<td>Sigma</td>
			<td>D9434</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethyl ether anhydrous grade</td>
			<td>Sigma</td>
			<td>296082</td>
			<td></td>
		</tr>
		<tr>
			<td>Drying oven</td>
			<td>Fisher</td>
			<td>11-475-155</td>
			<td></td>
		</tr>
		<tr>
			<td>EcoLume scintillation liquid</td>
			<td>VWR</td>
			<td>IC88247001</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf 5424R centrifuge</td>
			<td>Fisher</td>
			<td>05-401-205</td>
			<td></td>
		</tr>
		<tr>
			<td>GE Storage phosphor screen</td>
			<td>Sigma</td>
			<td>GE28-9564-75</td>
			<td></td>
		</tr>
		<tr>
			<td>GE Typhoon FLA9500 imager</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial acetic acid, ACS grade</td>
			<td>Sigma</td>
			<td>695092</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass 6mL scintillation vials</td>
			<td>Sigma</td>
			<td>M1901</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass centrifuge tube caps</td>
			<td>Fisher</td>
			<td>14-595-36A</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass centrifuge tubes</td>
			<td>Fisher</td>
			<td>14-595-35A</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Pasteur pipette</td>
			<td>Fisher</td>
			<td>13-678-20C</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexane, anhydrous grade</td>
			<td>Sigma</td>
			<td>296090</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Adenine &#62;99%</td>
			<td>Sigma</td>
			<td>A8626</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Alanine &#62;98%</td>
			<td>Sigma</td>
			<td>A7627</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Arginine &#62;99%</td>
			<td>Sigma</td>
			<td>A1270000</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Asparagine &#62;98%</td>
			<td>Sigma</td>
			<td>A0884</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Aspartate &#62;98%</td>
			<td>Sigma</td>
			<td>A9256</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine &#62;97%</td>
			<td>Sigma</td>
			<td>W326305</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamic acid monosodium salt monohydrate &#62;98%</td>
			<td>Sigma</td>
			<td>49621</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine &#62;99%</td>
			<td>Sigma</td>
			<td>G3126</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glycine &#62;99%</td>
			<td>Sigma</td>
			<td>G8898</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Histidine &#62;99%</td>
			<td>Sigma</td>
			<td>H8000</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Isoleucine &#62;98%</td>
			<td>Sigma</td>
			<td>I2752</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Leucine &#62;98%</td>
			<td>Sigma</td>
			<td>L8000</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Lysine &#62;98%</td>
			<td>Sigma</td>
			<td>L5501</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Methionine, HPLC grade</td>
			<td>Sigma</td>
			<td>M9625</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Phenylalanine, reagent grade</td>
			<td>Sigma</td>
			<td>P2126</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Proline &#62;99%</td>
			<td>Sigma</td>
			<td>P0380</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Serine &#62;99%</td>
			<td>Sigma</td>
			<td>S4500</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Theronine, reagent grade</td>
			<td>Sigma</td>
			<td>T8625</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Tryptophan &#62;98%</td>
			<td>Sigma</td>
			<td>T0254</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Tyrosine &#62;98%</td>
			<td>Sigma</td>
			<td>T3754</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Uracil &#62;99%</td>
			<td>Sigma</td>
			<td>U0750</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Valine &#62;98%</td>
			<td>Sigma</td>
			<td>V0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol, ACS grade</td>
			<td>Fisher</td>
			<td>A412</td>
			<td></td>
		</tr>
		<tr>
			<td>Oleic acid &#62;99%</td>
			<td>Sigma</td>
			<td>O1008</td>
			<td></td>
		</tr>
		<tr>
			<td>p-anisaldehyde</td>
			<td>Sigma</td>
			<td>A88107</td>
			<td></td>
		</tr>
		<tr>
			<td>Petroleum ether, ACS grade</td>
			<td>Sigma</td>
			<td>184519</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphatidylcholine, dipalmitoyl &#62;99%</td>
			<td>Sigma</td>
			<td>P1652</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes</td>
			<td>Eppendorf</td>
			<td>2231000713</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride, ACS grade</td>
			<td>Sigma</td>
			<td>P3911</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide pellets, certified ACS</td>
			<td>Fisher</td>
			<td>S318-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Squalene &#62;98%</td>
			<td>Sigma</td>
			<td>S3626</td>
			<td></td>
		</tr>
		<tr>
			<td>Succinic Acid crystalline/certified</td>
			<td>Fisher</td>
			<td>110-15-6</td>
			<td></td>
		</tr>
		<tr>
			<td>TLC saturation pad</td>
			<td>Sigma</td>
			<td>Z265225</td>
			<td></td>
		</tr>
		<tr>
			<td>TLC silica gel 60G glass channeled plate</td>
			<td>Fisher</td>
			<td>NC9825743</td>
			<td>No fluorescent indicators</td>
		</tr>
		<tr>
			<td>Transparency plastic film</td>
			<td>Apollo</td>
			<td>829903</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricine</td>
			<td>Sigma</td>
			<td>T0377</td>
			<td></td>
		</tr>
		<tr>
			<td>Triolein &#62;99%</td>
			<td>Sigma</td>
			<td>T7140</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>Fisher</td>
			<td>02-215-414</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman exposure cassette</td>
			<td>Sigma</td>
			<td>WHA29175523</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast nitrogen base without ammonium sulfate and amino acids</td>
			<td>Sigma</td>
			<td>Y1251</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of neutral lipid synthesis in saccharomyces cerevisiae by metabolic labeling and thin layer chromatography

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de novo&#160;from acetyl-CoA and are primarily present in eukaryotes in the form of triglycerides (TGs) and sterol-esters (SEs). The enzymes responsible for the synthesis of NLs are highly conserved from Saccharomyces cerevisiae (yeast) to humans, making yeast a useful model organism to dissect the function and regulation of NL metabolism enzymes. While much is known about how acetyl-CoA is converted into a diverse set of NL species, mechanisms for regulating NL metabolism enzymes, and how mis-regulation can contribute to cellular pathologies, are still being discovered. Numerous methods for the isolation and characterization of NL species have been developed and used over decades of research; however, a quantitative and simple protocol for the comprehensive characterization of major NL species has not been discussed. Here, a simple and adaptable method to quantify the de novo&#160;synthesis of major NL species in yeast is presented. We apply 14C-acetic acid metabolic labeling coupled with thin layer chromatography to separate and quantify a diverse range of physiologically important NLs. Additionally, this method can be easily applied to study in vivo reaction rates of NL enzymes or degradation of NL species over time.

In this protocol, we have demonstrated that the labeling, detection, and quantification of NL species can be accomplished by 14C-acetic acid metabolic labeling. Major NL species can be separated in a solvent system of 50:40:10:1 (v/v/v/v%) Hexane:Petroleum ether:Diethyl ether:Acetic acid (Figure 1A,B). Phosphor imaging allows for visualization of labeled free fatty acid (FFA), triacylglycerol (TG), diacylglycerol (DG), cholesterol (Chol), and squalene (SQ) (<stron...

Watch this Scientific Journal Video about Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography at JoVE.com

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Here, a protocol is presented for the metabolic labeling of yeast with 14C-acetic acid, which is coupled with thin layer chromatography for the separation of neutral lipids.

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de ...

This technique uses instruments commonly found in most laboratory environments and allows for the analysis of several samples simultaneously. This method can provide key insights into how lipid metabolism enzymes are regulated in a cellular context and in different genetic backgrounds. Inoculate a colony from a yeast culture plate into 20 milliliters of SC media containing 2%dextrose and incubate at 30 degrees Celsius for overnight with shaking at 200 rpm.On the next day, dilute the culture in 50 milliliters of fresh media to a final OD of 0.2 and grow it for 24 hours until the stationary phase is reached. Prepare two 20 milliliter aliquots of quenching buffer for each sample and store them at minus 80 degrees Celsius. Simultaneously, prepare radiolabeling media by adding acetic acid sodium salt to dextrose free SC media at a final concentration of 10 microcurie per milliliter.Collect the cells by centrifuging at 4, 100 x g for 10 minutes. Remove the supernatant and wash the cell pellet once with 20 milliliters of dextrose free SC media. Collect the cells again by centrifuging at 4, 100 x g for five minutes and transfer them to a labeled two milliliter microcentrifuge tube using dextrous free media.Centrifuge the cells for another two minutes. Resuspend the cells in 500 microliters of dextrose free SC media and begin the radiolabeling period by quickly adding 500 microliters of radiolabeling media to each cell suspension. Incubate the tubes in a rotating incubator at 30 degrees Celsius for 20 minutes.Meanwhile, pre cool the centrifuge equipped for 50 milliliter conical tubes to minus 10 degrees Celsius and thaw one 20 milliliter aliquots of quenching buffer. Once the radiolabeling period has ended use a pipette to plunge the entire one milliliter sample into thawed quenching buffer on ice. Collect the cell pellet by spinning in a pre cooled centrifuge at 5, 000 x g for three minutes and add 20 milliliters of fresh cold quenching buffer.Vortex and shake the samples to fully resuspend them in quenching buffer and centrifuge the samples again to collect the cells. Once the cells are pelleted, thoroughly remove all quenching buffer from the samples by pouring off the supernatant and removing the excess with a pipette. Store the tubes at minus 80 degrees Celsius for further processing.Weigh 0.3 grams of acid washed glass beads for each sample and store them in two milliliter microcentrifuge tubes on ice. Remove the cell pellets from the minus 80 degree freezer and place them on ice. Then, add 350 microliters of methanol and 700 microliters of chloroform to each sample and resuspend the cells.Transfer them to micro centrifuge tubes containing pre-weighed glass beads. Lyse the cells by vortexing three times for one minute, with 30 second incubation on ice between agitations. Pour the cells into a 15 milliliter glass centrifuge tube and label the tube as tube A.Wash the microcentrifuge tube with one milliliter of methanol, vortex it, and pour the methanol into tube A.Add two milliliters of chloroform followed by 400 microliters of water to tube A for a final sample volume of 4.45 milliliters.Vortex the samples for one minute followed by a five minutes centrifugation at 1000 x g to clearly separate the aqueous and organic phases with cell debris lying at the interface. Using a glass pasture pipette, collect the organic phase into a new glass centrifuge tube labeled B and add one milliliter of one molar potassium chloride. Add one milliliter of methanol and two milliliters of chloroform to tube A and repeat the vortexing and centrifugation steps.Vortex and centrifuge tube B to separate the aqueous and organic phase. Remove the upper aqueous layer and collect the entire bottom organic layer into a four milliliter glass vial. Completely evaporate the solvent from the lipid extracts by drying under inert gas and preheat an oven to 145 degrees Celsius for heating the TLC plate.Determine relative amounts of radiolabel taken up by the cells before loading them onto the TLC plate. Reconstitute the sample lipids in 40 to 50 microliters of chloroform and methanol mixed at a one-to-one ratio by vortexing for five minutes. Prepare 101 milliliters of the mobile phase solvent in a glass graduated cylinder.Then, pour the solvent into a glass TLC chamber containing a 20 by 20 TLC saturation pad and a tight fitting lid. Prepare a channeled 20 by 20 silica gel 60G plate and mark a line 1.5 centimeters above the bottom of the plate using a pencil. Once the plate has been sufficiently heated and the TLC saturation pad is saturated with solvent, remove the TLC plate from the oven and immediately proceed to loading it.Using a pipette, spot five microliters of sample onto the origin of each lane located 1.5 centimeters above the bottom of the TLC plate and repeat loading until 20 to 40 microliters of sample has been loaded into each lane. Once the standard and the samples have been loaded, place the plate in the developing chamber and wait until the solvent has reached the top of the plate. When the plate is fully developed, remove it from the chamber and allow it to dry in the fume hood for 20 minutes.After the plate is dried, cover it with plastic film and place it in a developing cassette with an autoradiography screen. Remove the screen from the developing cassette and place it inside of a phosphor imager. Spray the TLC plate with p-Anisaldehyde reagent until the silica is saturated, then bake it in a 145 degree oven for five minutes or until bands have appeared.To quantify lipid species, scrape the silica gel containing individual lipid species and transfer them to glass scintillation vials. Add six milliliters of scintillation fluid and vortex vigorously until the silica band has been reduced to small pieces. Place the rack with the scintillation vials into a scintillation counter and count by adjusting the counting time to two minutes per vial.Phosphor imaging allows for visualization of autoradiogram of label-free fatty acid, triacylglycerol, diacylglycerol, cholesterol, and squalane. It was demonstrated that purified lipid species can be separated in this method and subsequently visualized by spraying of the TLC plate with p-Anisaldehyde reagent. Visualized species include all previously mentioned lipids in addition to sterile esters.By applying a 10 minute chase period in radiolabel-free media following the pulse, it was observed at the major pool of squalane disappeared and total cholesterol was elevated. Similarly, the appearance of diacylglycerol in the chase period correlated with a decrease in the free fatty acids signal. Prior to performing the full protocol, it is important to determine whether the cells will uptake the acetic acid radiolabel in the growth condition and genetic background of interest.

Research

JoVE Journal

Biochemistry

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