Purification LDs from mouse hepatocytes

From LipidomicsWiki

Isolation of hepatocytes

Solutions:
Buffer 1: 1 L Krebs-Henseleit buffer (KHB) (NaCl 115 mmol/l; NaHCO3 25 mmol/l; KCl 5,9 mmol/l; MgCl2 1,18 mmol/l; NaH2PO4 1,23 mmol/l; Na2SO4 1,2 mmol/l; CaCl2 1,25 mmol/l, glucose 6 mmol/l)

Buffer 2: 1 L KHB without Ca2+ and SO42- (NaCl 115 mmol/l; NaHCO3 25 mmol/l; KCl 5,9 mmol/l; MgCl2 1,18 mmol/l; NaH2PO4 1,23 mmol/l; glucose 6 mmol/l)

Collagenase Solution: Mix 150 ml of buffer 2 with 1,5 ml of 10 mmol CaCl2. Add 3 g BSA (Fraction V, Sigma-Aldich) and 30 mg collagenase type CLS II (?)

Buffer 1 and 2 as well as collagenase solution have been filter-sterilized. Buffer 1 and 2 are incubated in the CO2 incubator for saturation of the solutions with CO2. Before perfusion, buffer 2 and the collagenase solution are heated up to 37 °C.

Perfusion:
Tubings are washed with ethanol and flushed with PBS (phosphate buffered saline) before perfusion.

1. The liver of anesthetized mice is perfused thought the V. porta hepatis with buffer 2 for and afterwards with collagenase solution heated to 37°C (15 min each).
2. The liver is carefully removed and put to a petri dish, filled with 4-5 ml collagenase solution. In this solution the liver is cut into small pieces, pushed through a sieve (µM) and flushed with ice-cold buffer 1
3. The cell suspension is filtrated into a 50 ml Greiner tube through a 70 µm cell strainer.
4. The cell suspension is mixed with 40 % iodixanol solution (Axis Shield), that the final concentration of iodixanol is 17 %
5. A layer of 2 ml of buffer 1 is added on the top
6. Centrifugation is carried out for 20 min at 4 °C at 400g ( 1400 rpm, Beckmann CS-6R)
7. Allow the rotor to decelerate without the brake to protect the gradient
8. Discard the supernatant to get the hepatocyte cell pellets

Purification of lipid droplets

Materials:
Disruption buffer: 20 mM K-phosphate pH 7.4, 250 mM sucrose, protease inhibitor mix, 1 mM EDTA, 1 mM PMSF
Overlay buffer : 50 mM potassium phosphate pH 7.4, 100 mM KCl, 1 mM EDTA, 1 mM PMSF

1. Resuspend pellets in disruption buffer containing 1 mM PMSF as protease inhibitor and 1 mM EDTA

2. Cells are disrupted with a Schütt-Homogenisator, ice-cooling, 300 rpm, 8 strokes
For examination of disruption quality, the suspension is centrifuged for 5 min at 1000 g (Beckman CS-6R)

3. The cell suspension is transferred to a centrifuge tube (SW41) and overlayed with the overlay buffer.

4. Centrifugation is carried out at 4 °C for 1h, 100.000 g (= 40.000 rpm; Beckmann Ultrazentrifuge, Rotor = SW41) The lipid droplets concentrate in a white band at the top of the gradient.

5. Lipid droplets are collected with a pipette tip and transferred to another centrifuge tube, adjusted to 0.25M sucrose, and again overlaid with with the overlay buffer. Centrifugation is carried out at 4 °C for 1h, 100.000 g (= 40.000 rpm; Beckmann Ultrazentrifuge, Rotor = SW41).

6. Collect floating lipid droplets with a pipette tip with a minimum of buffer and transfer them to a tube.