Sphingolipids (glycosphingolipid, sphingomyelin, ceramide, sphingosine and sphingosine 1-phosphate), defined as lipids containing a sphingoid base, are ubiquitously distributed in the bio-membranes of mammals and invertebrates including some microbes. Metabolic labeling of sphingolipids is usually performed using a radioactive precursor of sphingolipid synthesis such as [14C]serine or [14C]/ [3H]sphingosine. In the case of glycosphingolipids, [14C]galactose is also frequently used. This section deals with the metabolic labeling of sphingolipids in mammalian cell cultures and the yeast Saccharomyces cerevisiae. |
Category | Biosynthesis & Metabolism |
Protocol Name | Metabolic labeling of sphingolipids |
Authors
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Tani, Motohiro
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Department of Chemistry, Faculty of Sciences, Kyushu University
Ito, Makoto
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University
*To whom correspondence should be addressed.
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KeyWords |
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Reagents
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L-[U-14C]serine (150 mCi/mmol, PerkinElmer, Waltham, MA) |
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D-erythro-[3H]-sphingosine (15-30 Ci/mmol, PerkinElmer) |
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EN3HANCE spray (PerkinElmer) |
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Silica Gel 60 TLC plate (Merck Millipore, Billerica, MA) |
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MEM medium (Sigma-Aldrich, St. Louis, MO) |
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Ham’s F-12 medium (Sigma-Aldrich) |
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YPD medium (1% yeast extract, 2% peptone, and 2% glucose) |
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SC medium (0.67% yeast nitrogen base w/o amino acids (BD, Franklin Lakes, NJ), and 2% glucose) containing nutritional supplements (Tong and Boone, 2006) |
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Instruments
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Imaging plate (Fujifilm, Tokyo, Japan) |
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Imaging analyzer (FLA5000 model, Fujifilm) |
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Film processing system (Cepros Q model, Fujifilm) |
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Methods |
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Metabolic labeling of B16 melanoma cells with [14C]serine
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1) |
Pre-culture the B16 melanoma cells in minimum essential medium (MEM) supplemented with 10% FBS, 100 μg/mL of streptomycin, and 100 units/mL of penicillin in a CO2 incubator. |
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Culture the cells described in step 1-1) in a 6-well plate (1 × 105 cells/well) overnight. |
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Add 1 μCi of [14C]serine to each well. |
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Incubate at 37°C for 24 h in a CO2 incubator. |
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Harvest the cells using a cell scraper. |
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Centrifuge the cells at 700 × g for 5 min at 4°C, wash with ice-cold PBS,and then re-suspend in 750 μL of i-propanol, hexane, and water (55/35/10, v/v/v). |
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Sonicate the suspension for 20 min in a sonic bath. |
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Centrifuge at 10,000 × g for 3 min. |
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Withdraw the supernatant and transfer it to a new tube. |
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Dry the sample with a centrifugal concentrator. |
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Dissolve the dried material in 20 μL of chloroform and methanol (2/1, v/v). |
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Spot the sample on a Silica Gel 60 TLC plate and perfom two-dimensional TLC as described in step 1-13)~15). |
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Develop the TLC plate with chloroform, methanol, formic acid, and H2O (65/25/8.9/1.1, v/v/v/v) (1st run). |
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Dry the TLC plate with a hair-dryer. Rotate it 90° and develop with chloroform, methanol, and 5 N NH4OH (50/40/10, v/v/v) (2nd run). |
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Dry the TLC plate with a hair-dryer. Develop the plate with diethylether in the opposite direction to the second run to separate ceramides from other neutral lipids. |
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Dry the TLC plate with a hair-dryer. Put the TLC plate onto an imaging plate of the exposure cassette. |
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17) |
Expose the plate overnight and use an imaging analyzer. |
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2. |
Metabolic labeling of CHO cells with [3H]sphingosine
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1) |
Culture the CHO cells in Ham’s F-12 medium supplemented with 10% FBS, 100 μg/mL of streptomycin, and 100 units/mL of penicillin in a CO2 incubator. |
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Culture the cells described in step 2-1) in a 6-well plate (1 × 105 cells/well) overnight. |
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Add 0.5 μCi of [3H]sphingosine to each well. |
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Incubate at 37°C for 24 h in a CO2 incubator. |
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Harvest the cells using a cell scraper. |
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Centrifuge at 700 × g for 5 min at 4°C, wash with ice-cold PBS, and then re-suspend in 100 μL of ice-cold water. |
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Add 375 μL of chloroform, methanol, and HCl (100/200/1, v/v/v) to the cell suspension, and mix well with a Voltex mixer. |
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Add 125 μL of chloroform and 125 μL of 1% KCl, and mix well with a Voltex mixer. |
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Centrifuge at 10,000 × g for 5 min, withdraw the lower phase (organic phase), and transfer it to a new tube. |
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Dry the sample with a centrifugal concentrator. |
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11) |
Dissolve the sample in 20 μL of chloroform and methanol (2/1, v/v). |
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12) |
Spot the sample on a Silica Gel 60 TLC plate and develop it with 1-butanol, acetic acid, and water (3/3/1, v/v/v). |
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13) |
Dry the TLC plate with a hair-dryer and spray the TLC with an enhancer EN3HANCE. |
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14) |
Expose the TLC plate to X-ray film for at least 2 days at –80°C. Visualize the radioactive spot with a film processing system. |
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3. |
Metabolic labeling of yeast Saccharomyces cerevisiae with [14C]serine
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1) |
Culture the yeast cells overnight in YPD medium. |
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Dilute the culture with SC medium lacking serine to 0.5 A 600 units/mL and culture for an additional 5 h. |
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Collect the cells by centrifugation at 1,500 × g for 3 min, and re-suspend in 500 μL of fresh SC medium lacking serine to 2.0 A600 units/mL. |
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Add 0.5 μCi of [14C]serine to the cell culture. |
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Incubate for 1 h at 30°C with gentle shaking. |
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Harvest the cells by centrifugation at 1,500 × g for 3 min at 4°C and then wash with 1 mL of ice-cold water. |
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Add 150 μL of ethanol, water, diethyl ether, pyridine, and 15 N ammonia (15/15/5/1/0.018, v/v/v/v/v) to the cell pellets. |
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Mix the sample with a Voltex mixer. |
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Centrifuge at 10,000 × g for 1 min and withdraw the supernatant. |
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Add the solvent to the residue and extract the sphingolipids again as described in step 3-7). |
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Mix the supernatant obtained in step 3-10) and 11). |
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13) |
Dry the supernatant with a centrifugal concentrator. |
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14) |
Dissolve the dried sample in 20 μL of chloroform, methanol, and water (5/4/1, v/v/v). |
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15) |
Spot the sample on a Silica Gel 60 TLC plate and then develop with chloroform, methanol, and 4.2 N ammonia (9/7/2, v/v/v). |
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16) |
Dry the TLC plate, expose it to an imaging plate, and then use an imaging analyzer. |
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Figure & Legends |
Figure & Legends
Fig.1. Radio-labeling of B16 melanoma cells with [14C]serine
Fig. 2. Radio-labeling of CHO cells with [3H]sphingosine
Fig. 3. Radio-labeling of yeast Saccharomyces cerevisiae with [14C]serine
The extracted lipids were subjected to mild alkaline treatment with monomonomethyamine (MMA). |
Copyrights |
Attribution-Non-Commercial Share Alike
This work is released underCreative Commons licenses
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Date of registration:2014-11-21 09:32:05 |
- Komori, H., Ichikawa, S., Hirabayashi, Y., and Ito, M. (1999) Regulation of intracellular ceramide content in B16 melanoma cells. Biological implications of ceramide glycosylation. J Biol Chem. 274, 8981–8987 [PMID : 10085144]
- Tong, A.H., and Boone, C. (2006). Synthetic genetic array analysis in Saccharomyces cerevisiae. Methods Mol Biol. 313, 171–192 [PMID : 16118434]
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Metabolic labeling of sphingolipids.
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Ito, Makoto,
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