Triglyceride hydrolysis kinetics

From LipidomicsWiki

Assay of triglyceride hydrolysis kinetics in cultured cells (A431, HeLa)

1. Split the cells on 35mm dishes or 6-well plates.
2. The next day label and load cells by adding 0.8ml 3H-oleic acid-oleic acid-BSA complexes (see instruction below) overnight in serum containing growth medium.
3. After the labeling wash cells twice with cold PBS.
4. Add the chase medium to the cells [containing 5% delipidated serum and 6µM Triacsin C (the latter optional)].
5. After time points (for example 0, 4, 10, 24h) wash the cells 2x with PBS and harvest cells to 2% NaCl (cold).
6. Pellet cells 1000xg 10min +4ºC.
7. Remove the supernatants and freeze the cell pellets in -70ºC.
8. Suspend the cells to 0.9ml 2%NaCl.
9. Take 100µl samples for the protein determination. Add 5µl 20% SDS to the protein samples and incubate 1hr +37ºC before the protein assay.
10. Transfer the 800µl samples to capped class tubes.
11. Bligh and Dyer extraction: Add 2 ml methanol and 1 ml chloroform, vortex.
12. Centrifuge 1000xg 10 min.
13. Transfer the supernatants to fresh tubes.
14. Add 1 ml water and 1 ml chloroform, vortex.
15. Centrifuge 1000xg 10 min.
16. Transfer the lower phase to a fresh tube (You can freeze the samples in -20°C).
17. Evaporate the samples with nitrogen gas.
18. Dissolve in 40µl chloroform:methanol 9:1.
19. 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 a triolein standard.
20. Run the TLC with 65:15:1:0.25 hexane:diethylether:acetic acid:water. The run takes approximately 30-40 minutes.
21. Dry the plate and stain in iodine tank.
22. Mark the spots with the help of the standard and scrape to scintillation tubes.
23. Analyze 3H radioactivity by liquid scintillation counting.
24. Determine the protein concentration of the frozen samples with Bio-Rad DC -kit and correct the scintillation results with the protein amounts.

Making oleic acid-BSA complexes in culture medium
(1mM oleic acid in 0.5% BSA)

Ref. Spector, A. A. (1986) Structure and lipid binding properties of serum albumin. Methods Enzymol 128, 320-339.

1. Dissolve 0.125g bovine serum albumin (fatty acid free; MP Biochemicals cat. no. 105033) in 25ml medium with no additions in the 37ºC water bath.
2. Dissolve 30 mg oleic acid (sodium salt; Sigma cat. no. 0-7501) in 1ml water in 37ºC (final conc. 100mM).
3. Adjust the BSA-medium to pH 10 with NaOH.
4. Add 250µl oleic acid dropwise to BSA-medium while vortexing. Add slowly.
5. Add [9,10(n)-3H]oleic acid (Amersham TR140, 7Ci/mmol; 6.67µCi/µl) 1:1000 (final conc. 6.7µCi/ml).
6. Immediately adjust to pH 7.7 with HCl.
7. Filter sterilize with 0.22µm filters.
8. Store at +4ºC for up to 2 weeks.