Biology Reference
In-Depth Information
FIGURE 10.4
Lipid droplets staining in HCV-infected cells. Representative images (scale bar
10 mm)
of Huh7-Lunet cells transfected with HCV JFH1 RNA. Cells were fixed and stained with anti-
core antibodies, and Bodipy 493, or Neutral LipidTox Red, or Neutral LipidTox Far red, or
ORO and Hoechst (nucleus, blue). Cells were fixed with 0.2% Triton X-100 for 10 min,
washed three times with PBS, and incubated with blocking solution (DMEM with 5% FBS
and 50% goat serum) for 20 min. Cell were washed three times and then incubated with
primary antibodies (anti-Core, Sigma) in PBS with 1% BSA for 1 h, washed, and then
incubated with secondary antibodies (anti-mouse Alexa 595 or Alexa 488 (Invitrogen)) in
PBS plus 1% BSA. Cells were then stained for Lipid Droplets, and mounted with DAPI
(Vectashield). Cells were analyzed by epifluorescence microscopy.
ΒΌ
10.2.2.5
Quantification
Quantification of LDs per cell or colocalization of a protein with LDs can be mea-
sured using the open source ImageJ program, or by using the automatic measurement
features in programs, such as Volocity (Perkin Elmer) or AxioVision (Zeiss).
10.2.3
Lipid measurements
Viral infection can cause changes in cell lipid metabolism. This has been reported
mainly for HCV and DENV. In addition to quantification of LDs by microscopy,
