Isolation of lipid droplets from mouse primary hepatocytes

From LipidomicsWiki

Solutions

Disruption buffer: 20 mM Potassium phosphate pH 7.4, 250 mM sucrose, 1 mM EDTA, 1 mM PMSF.

Overlay buffer: 50 mM Potassium phosphate pH 7.4, 100 mM KCl, 1 mM EDTA, 1
mM PMSF.

Sucrose (1 M), ice cold

Purification

1. Re-suspend hepatocyte pellet (originating from livers of 3 mice) in 5 ml ice cold disruption buffer.

2. For N2-cavitation of hepatocytes fill cell suspension into N2-bomb (45 ml, Parr Instruments), cooled on crashed ice, and apply N2-pressure of 800 psi for 5 min.

3. Place homogenate into 50 ml Greiner-tube and centrifuge at 1000 g (Beckman CS-6R rotor) for 5 min at 4 °C to remove cell debris.

4. Transfer homogenate is into a ultracentrifuge tube (SW41) and add ice cold overlay buffer up to ~3 mm below tube top.

4. Centrifugation is carried out at 100.000 g (40.000 rpm, Beckman Ultracentrifuge, SW41 rotor) for 1 h at 4 °C, lipid droplets concentrate in a white band at the top of gradient.

5. Two ml lipid droplets are collected with a pipette tip and transferred to another SW41 centrifuge tube; sucrose solution (1 M) is added to arrive at a 0.25 M sucrose concentration. Then add again overlay buffer up to around 3 mm below tube top.