Purification LDs from mouse adipocytes

From LipidomicsWiki

Purification of lipid droplets from adipocytes

1. Resuspend isolated fat cells in disruption buffer containing protease inhibitors and 1 mM EDTA.

2. Disrupt cells with a nitrogen bomb.
For nitrogen cavitation, apply 800 psi for 5 min on ice (max vol 2ml).
Adjust sucrose concentration with 1 vol of 1.08M sucrose in disruption buffer
Centrifuged at 1000 g (10 min, 4°C) to remove nuclei.
3. Purify lipid droplets on a sucrose gradient.
Transfer the supernatant fraction to a SW41 centrifuge tube
Overlay with disruption buffer containing decreasing sucrose (from 0.25M to 0).
The gradient is centrifuged for 1hour at 150.000 xg, 10°C.
The lipid droplets concentrate in a white band at the top of the gradient.

4. Collect lipid droplet fraction with a pipette tip trying to obtain the most lipid droplets with the least amount of buffer, and transfer in an Eppendorf tube.
If needed Lipid droplets are separated from buffer by centrifuging at 20,000 xg for 5 minutes 4°C. The underlying solution is removed.

Materials :
Disruption buffer without sucrose: 25 mM Tris pH7.4, 100m M KCl, 5 mM EGTA.
Cell disruption bomb: PARR Instrument company. Ref 4639
SW41 rotor