Free cholesterol/cholesterol ester distribution

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Current revision (12:21, 26 April 2010) (view source)
 
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1. In the morning, split cells (A431, HeLa, CHO) on 6-well plates in complete medium,volume 2ml<br>2. In the afternoon add 2µl/well (0.1µCi/ml) [14C]cholesterol (Amersham CFA128, 58mCi/mmol) <br>3. Label the cells for 40h in +37ºC (after this you can optionally equilibrate the radioactive cholesterol with the cellular pools for 24 h)<br>4. Wash the cells two times with PBS <br>5. Harvest the cells to 2% NaCl<br>6. Pellet cells 1000xg 10min in +4ºC. You can freeze the pellets at this point.<br>7. Suspend the cells in 0.9ml 2%NaCl<br>8. Take 100µl samples for protein determination. Add 5µl 20% SDS to the protein samples and incubate 1hr +37ºC before the protein assay (BioRad DC assay).<br>9. Transfer the 800µl samples to capped class tubes.<br>10. Bligh and Dyer extraction: Add 2 ml methanol and 1 ml chloroform, vortex.<br>11. Centrifuge 1000xg 10 min.<br>12. Transfer the supernatants to fresh tubes.<br>13. Add 1 ml water and 1 ml chloroform, vortex.<br>14. Centrifuge 1000xg 10 min.<br>15. Transfer the lower phase to a fresh tube (You can here freeze the samples in -20°C).<br>16. Evaporate the samples with nitrogen gas.<br>17. Dissolve in 40µl chloroform:methanol 9:1<br>18. Apply the samples (using a Hamilton syringe) on TLC plates (Merck silica gel 60, cat. no. 1.05721.0001) in a line with the length of 1 cm (1.5 cm between the samples). Pipet also cholesterol and cholesteryl ester standards<br>19. Run the TLC with 60:40:1 petrolether:diethylether:acetic acid. The run takes approximately 30-40 minutes.<br>20. Dry the plate and stain in iodine tank.<br>21. Mark the cholesterol and cholesteryl ester spots with the help of the standards and scrape into scintillation tubes.<br>22. Analyze radioactivity by liquid scintillation counting.<br>23. The results are expressed as percentage of free cholesterol and cholesteryl esters of total cholesterol or as DPM/mg protein.<br>  
1. In the morning, split cells (A431, HeLa, CHO) on 6-well plates in complete medium,volume 2ml<br>2. In the afternoon add 2µl/well (0.1µCi/ml) [14C]cholesterol (Amersham CFA128, 58mCi/mmol) <br>3. Label the cells for 40h in +37ºC (after this you can optionally equilibrate the radioactive cholesterol with the cellular pools for 24 h)<br>4. Wash the cells two times with PBS <br>5. Harvest the cells to 2% NaCl<br>6. Pellet cells 1000xg 10min in +4ºC. You can freeze the pellets at this point.<br>7. Suspend the cells in 0.9ml 2%NaCl<br>8. Take 100µl samples for protein determination. Add 5µl 20% SDS to the protein samples and incubate 1hr +37ºC before the protein assay (BioRad DC assay).<br>9. Transfer the 800µl samples to capped class tubes.<br>10. Bligh and Dyer extraction: Add 2 ml methanol and 1 ml chloroform, vortex.<br>11. Centrifuge 1000xg 10 min.<br>12. Transfer the supernatants to fresh tubes.<br>13. Add 1 ml water and 1 ml chloroform, vortex.<br>14. Centrifuge 1000xg 10 min.<br>15. Transfer the lower phase to a fresh tube (You can here freeze the samples in -20°C).<br>16. Evaporate the samples with nitrogen gas.<br>17. Dissolve in 40µl chloroform:methanol 9:1<br>18. Apply the samples (using a Hamilton syringe) on TLC plates (Merck silica gel 60, cat. no. 1.05721.0001) in a line with the length of 1 cm (1.5 cm between the samples). Pipet also cholesterol and cholesteryl ester standards<br>19. Run the TLC with 60:40:1 petrolether:diethylether:acetic acid. The run takes approximately 30-40 minutes.<br>20. Dry the plate and stain in iodine tank.<br>21. Mark the cholesterol and cholesteryl ester spots with the help of the standards and scrape into scintillation tubes.<br>22. Analyze radioactivity by liquid scintillation counting.<br>23. The results are expressed as percentage of free cholesterol and cholesteryl esters of total cholesterol or as DPM/mg protein.<br>  
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[[Category:Lipid metabolism analysis]]
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[[Category:Lipid_metabolism_analysis]] [[Category:Radioactive_labeling]]

Current revision

Free cholesterol/cholesterol ester distribution using [14C]cholesterol labeling

1. In the morning, split cells (A431, HeLa, CHO) on 6-well plates in complete medium,volume 2ml
2. In the afternoon add 2µl/well (0.1µCi/ml) [14C]cholesterol (Amersham CFA128, 58mCi/mmol)
3. Label the cells for 40h in +37ºC (after this you can optionally equilibrate the radioactive cholesterol with the cellular pools for 24 h)
4. Wash the cells two times with PBS
5. Harvest the cells to 2% NaCl
6. Pellet cells 1000xg 10min in +4ºC. You can freeze the pellets at this point.
7. Suspend the cells in 0.9ml 2%NaCl
8. Take 100µl samples for protein determination. Add 5µl 20% SDS to the protein samples and incubate 1hr +37ºC before the protein assay (BioRad DC assay).
9. Transfer the 800µl samples to capped class tubes.
10. Bligh and Dyer extraction: Add 2 ml methanol and 1 ml chloroform, vortex.
11. Centrifuge 1000xg 10 min.
12. Transfer the supernatants to fresh tubes.
13. Add 1 ml water and 1 ml chloroform, vortex.
14. Centrifuge 1000xg 10 min.
15. Transfer the lower phase to a fresh tube (You can here freeze the samples in -20°C).
16. Evaporate the samples with nitrogen gas.
17. Dissolve in 40µl chloroform:methanol 9:1
18. Apply the samples (using a Hamilton syringe) on TLC plates (Merck silica gel 60, cat. no. 1.05721.0001) in a line with the length of 1 cm (1.5 cm between the samples). Pipet also cholesterol and cholesteryl ester standards
19. Run the TLC with 60:40:1 petrolether:diethylether:acetic acid. The run takes approximately 30-40 minutes.
20. Dry the plate and stain in iodine tank.
21. Mark the cholesterol and cholesteryl ester spots with the help of the standards and scrape into scintillation tubes.
22. Analyze radioactivity by liquid scintillation counting.
23. The results are expressed as percentage of free cholesterol and cholesteryl esters of total cholesterol or as DPM/mg protein.

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