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Metabolic labeling of sphingolipids
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Metabolic labeling of sphingolipids

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Introduction Protocol References Credit lines
Category
Biosynthesis & Metabolism
Protocol Name

Metabolic labeling of sphingolipids

Authors
Tani, Motohiro *
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.
KeyWords
Reagents

L-[U-14C]serine (150 mCi/mmol, PerkinElmer, Waltham, MA)

D-erythro-[3H]-sphingosine (15-30 Ci/mmol, PerkinElmer)

EN3HANCE spray (PerkinElmer)

Silica Gel 60 TLC plate (Merck Millipore, Billerica, MA)

MEM medium (Sigma-Aldrich, St. Louis, MO)

Ham’s F-12 medium (Sigma-Aldrich)

YPD medium (1% yeast extract, 2% peptone, and 2% glucose)

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)

Instruments

Imaging plate (Fujifilm, Tokyo, Japan)

Imaging analyzer (FLA5000 model, Fujifilm)

Film processing system (Cepros Q model, Fujifilm)

Methods
1.

Metabolic labeling of B16 melanoma cells with [14C]serine

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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2) 

 Culture the cells described in step 1-1) in a 6-well plate (1 × 105 cells/well) overnight.

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3) 

 Add 1 μCi of [14C]serine to each well.

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4) 

 Incubate at 37°C for 24 h in a CO2 incubator.

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5) 

 Harvest the cells using a cell scraper.

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6) 

 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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7) 

 Sonicate the suspension for 20 min in a sonic bath.

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8) 

 Centrifuge at 10,000 × g for 3 min.

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9) 

 Withdraw the supernatant and transfer it to a new tube.

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10) 

 Dry the sample with a centrifugal concentrator.

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11) 

 Dissolve the dried material 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 perfom two-dimensional TLC as described in step 1-13)~15).

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13) 

 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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14) 

 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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15) 

 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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16) 

 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

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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2) 

 Culture the cells described in step 2-1) in a 6-well plate (1 × 105 cells/well) overnight.

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3) 

 Add 0.5 μCi of [3H]sphingosine to each well.

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4) 

 Incubate at 37°C for 24 h in a CO2 incubator.

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5) 

 Harvest the cells using a cell scraper.

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6) 

 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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7) 

 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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8) 

 Add 125 μL of chloroform and 125 μL of 1% KCl, and mix well with a Voltex mixer.

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9) 

 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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10) 

 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

1) 

 Culture the yeast cells overnight in YPD medium.

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2) 

 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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3) 

 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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4) 

 Add 0.5 μCi of [14C]serine to the cell culture.

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5) 

 Incubate for 1 h at 30°C with gentle shaking.

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6) 

 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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7) 

 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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8) 

 Mix the sample with a Voltex mixer.

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9) 

 Incubate at 60°C for 15 min.

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10) 

 Centrifuge at 10,000 × g for 1 min and withdraw the supernatant.

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11) 

 Add the solvent to the residue and extract the sphingolipids again as described in step 3-7).

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12) 

 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).

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