Isolation of Lipid Rafts from Cultured Mammalian Cells and Their Lipidomics Analysis

Part I: Isolation of Lipid Rafts

The protocol described below was used to isolate rafts from RAW 264.7 murine macrophages, THP-1 human macrophages and SH-SY5Y human neuroblastoma cells, but is applicable to most cultured mammalian cells.

Materials and Reagents

1. 12 ml ultracentrifuge tubes (Beckman Coulter, catalog number: 344059 )
2. 1.5 ml Eppendorf Safe-Lock Tube
3. Spinal needle (BD, catalog number: 405256 )
4. 75 cm² flask
5. Fetal bovine serum (FBS)
6. OptiPrep^TM Density Gradient Medium (iodixanol solution, Sigma-Aldrich, catalog number: D1556-250ML )
7. Complete Protease inhibitors cocktail tablets (Roche, catalog number: 0 4906837001 )
8. Complete Phosphatase inhibitors cocktail tablets (Roche, catalog number: 0 4693124001 )
9. Sodium chloride (NaCl)
10. Potassium chloride (KCl)
11. Na₂HPO₄·2H₂O
12. KH₂PO₄
13. Tris hydrochloride (Tris-HCl)
14. Ethylenediaminetetraacetic acid (EDTA)
15. Triton X-100
16. Sucrose
17. Phosphate buffered saline (PBS; pre-made or see Recipes)
18. RPMI media
19. PBS (ice cold) (see Recipes)
20. Solution A (see Recipes)
21. Solution B (diluent) (see Recipes)
22. Solution C (working solution) (see Recipes)
23. Solution D (resuspension medium 1) (see Recipes)
24. Solution E (resuspension medium 2) (see Recipes)
    

Equipment

1. Pipettes
2. Centrifuge (Beckman Coulter, model: Allegra X-15R )
3. Ultracentrifuge (Beckman Coulter, model: Optima MAX-TL )
4. Ultracentrifuge (Beckman Coulter, model: Optima L-90K )
5. Two-chamber gradient former (Sigma-Aldrich, catalog number: Z340391 )
6. Misonix sonicator S-4000 with micro tip
7. Swing bucket rotor (SW41 Ti or similar)
8. CO₂ incubator
9. -80 °C freezer
   

Procedure