Biology Reference
In-Depth Information
10 mM oleic acid coupled to BSA (10 mg/ml)
Bodipy 558/568 C12 fatty acid analogue (1 mg/ml in ethanol, Life Technologies)
Lipofectamine 2000 (Life Technologies)
Confocal microscope with photobleaching module
14.2.1.2
Experimental procedures
14.2.1.2.1
Cell culture and metabolic labeling
1.
Seed 2 ml 3T3-L1 preadipocytes in the 35 mm Labtek culture dish at
0.2
10
6
cell/ml concentration.
2.
After overnight culture, cells are transfected with specific plasmid or siRNA
with Lipofectamine 2000 according to the manufacturer's instruction.
3.
4-6 h after transfection, 3T3-L1 preadipocytes are washed with
DMEM several times and incubated in fresh DMEM containing 10% FBS,
200
m
M oleic acids, and 0.2-1
m
g/ml Bodipy 558/568 C12 fatty acids for
14-18 h.
4.
For differentiated 3T3-L1 adipocytes, cells are incubated in similar medium
containing 5
m
g/ml insulin and 0.2-1
m
g/ml Bodipy 558/568 C12 fatty acid for
24 h before experiment.
14.2.1.2.2
FRAP setting
5.
1 h before FRAP experiment, cells are washed with prewarmed PBS buffer and
incubated in DMEM containing 10% FBS.
6.
Cells are then incubated in the live cell imaging chamber of microscope for at
least 15 min at 37
C with 5% CO
2
before photobleaching.
7.
Set microscope to 63
oil immersion objective; set pinhole at 1.0 air unit.
8.
Select a single LD under the microscope. Select a circular bleaching area
with 1-1.5
m diameter in the LD center. Note that the diameter of the
bleaching area should not exceed one-third of the LD diameter to ensure only
the selected LD is bleached.
9.
Find the optimal bleaching laser power and bleaching iteration number to allow
approximately 80% fluorescent bleaching efficiency within 1 min bleaching
duration.
10.
Set the time-lapse interval to be 20 s. Under situation that fluorescent signal in
the bleached LD can be recovered within a short period of time, repeated
bleaching cycles can be performed to collect multiple sets of data from a single
LD pair (see
Fig. 14.2
, middle panel).
11.
Set the imaging laser power and digital gain to ensure that there is no
overexposure of Bodipy dye fluorescence.
m
14.2.1.2.3
Data collection
12.
Identify cells that contain LD pairs in close apposition. For 3T3-L1
preadipocytes that are transfected with Fsp27-GFP, closely contacted LD pair
can be easily identified by the presence of intense GFP green fluorescent signal
