Biology Reference
In-Depth Information
4.
20-24 h after transfection, cells are washed with prewarmed PBS buffer and
fixed in 4% paraformaldehyde in PBS for 1 h at room temperature.
5.
Fixed cells on cover slip are washed in PBS buffer and stained
with 500 nM DAPI in PBS for 5 min. Cover slips are then mounted with
mounting media.
6.
LD morphology from transfection positive cells is recorded using confocal
microscope or any microscope equipped with fluorescence and optical
sectioning (e.g., ApoTome, Carl Zeiss).
7.
For LD sizemeasurement, Bodipy 558/568 channel of the images is further analyzed
in ImagePro software. Press “Menu”-
“Count/Size mode” and
select “Automatic Bright Objects.” In case the programcould not identify individual
LD due to its clustering, use “Edit”-
>
“Measure”-
>
>
“Watershed Split” followed by manual
“Split Objects.” During the analysis, visual and manual
inspection is needed to confirm that all LDs are identified.
8.
Select Diameter (Mean) in Count/Size-
splitting by using “Edit”-
>
>
Measure-
>
Select Measurements and
measure the diameters of all identified LDs.
9.
Export the measurement data into Excel for calculation of LD size distribution.
Images from approximately 30 cells are required for each distribution histogram
(
Fig. 14.1
).
10.
For measuring the largest LD in Fsp27-positive cells, over 300 positive cells
are selected and the size of the largest LD in each cell is extracted from the LD
size data. Similar method can be used in any adherent cells. Alternatively,
counting cells that contain LDs larger than a threshold diameter can also be used
to describe LD growth. This analysis will help overcome the heterogeneity
of cell population. We have defined large LDs as those having 2.5
min
diameter in Fsp27-GFP positive 3T3-L1 preadipocytes and calculated the
percentage of cells containing large LDs. These two types of analysis provide
simple, quantitative, and reliable ways to quantify LD sizes.
m
14.1.2
Data analysis and representative results
LD sizes are systematically measured in 3T3-L1 preadipocytes that express Fsp27
alone or Fsp27 and Plin1 using the method described above to measure the size dis-
tribution. In control 3T3-L1 preadipocytes that do not express Fsp27 or Plin1, ma-
jority of the LDs have diameters below 1
m. Ectopic expression of Fsp27-GFP leads
to a dramatic increase in LD sizes with diameter larger than 2
m
m and reduced LD
number (
Fig. 14.1
). Importantly, coexpression of Fsp27 and Plin1 further increase
the LD population with diameters between 3 and 6.5
m
m(
Fig. 14.1
).
m
14.2
MEASURING NEUTRAL LIPID EXCHANGE THROUGH FRAP
Neutral lipid exchange via LDCS is the hallmark of Fsp27-mediated LD fusion and
growth. The rate of neutral lipid exchange at LDCS can be conveniently and quan-
titatively measured by fluorescence recovery after photobleaching (FRAP) on
metabolically labeled LDs and calculated by the algorithm we have developed.
