Biomedical Engineering Reference
In-Depth Information
3. After 24 h of transfection, remove the culture medium and
wash the cells twice with cold PBSCM.
4. For cell fi xation: add 2.0 ml of cold 3 % paraformaldehyde to
each well of the plate, and keep the cells at 4 °C for 30 min.
5. For cell permeabilization, wash the cells thrice with PBSCM
containing 50 mM NH
4
Cl followed by two washes with
PBSCM (1 min per wash), then add 2.0 ml of 0.1 % saponin to
each well of the plate at room temperature for 15 min to per-
meabilize the cells (
see
Note 5
).
6. For labeling the lipid droplets: Remove the coverslip to a new
plate. Incubate the coverslip with 100
l of Oil Red O, Nile
Red, or BODIPY 493/503 dyes for each staining for 20 min
at room temperature, respectively, with the face seeded with
cells down immersed in the staining dyes.
7. After staining, wash the cells thrice with PBSCM (3 min per
wash).
8. Mount the coverslip on a glass slide with VECTASHIELD
mounting buffer with the face seeded cells down. Seal the cov-
erslip with nail polish.
9. The labeled cells are analyzed with Carl Zeiss LSM5 EXITER
laser scanning confocal microscope. GFP-Rab40c is viewed
directly under GFP channel in microscope, while lipid droplets
are revealed by the dyes Oil Red or Nile Red (Fig.
1
).
ʼ
3.2 Lipid Droplets
Fractionation by
Sucrose Density-
Gradient
Centrifugation
1. Grow 7-10 cm dishes of HepG2 cells in DMEM as mentioned
above.
2. Replace the medium with induction medium (refer to
Subheading
2.7
) until the culture reach 80 % of confl uence,
and grow the cells for another 24 h.
3. Wash the cells twice with 10 ml ice-cold PBS per dish
4. Scrape the cells into a 15 ml falcon tube with 2 ml disruption
buffer. Keep the cells on ice 30 min, and then disrupt the cells
by passing the cell suspension through a 27# gauge needle (
see
Note 6
).
5. Remove the cell debris by spinning at 1,000 ×
g
for 5 min at
4 °C.
6. Adjust the sucrose concentration of the supernatant to 0.33 M
and the fi nal volume to 3.0 ml (
see
Note 7
).
7. To make the sucrose density-gradient, 3.0 ml of cell lysate with
0.33 M of sucrose layered in the bottom of the ultracentrifuge
tube for SW41Ti rotor, followed by 3 ml of 0.25 M, 3 ml of
0.125 M sucrose solution, and 3 ml of disruption buffer.
8. Run the centrifuge at 150,000 ×
g
at 4 °C for 2 h.
