Biomedical Engineering Reference
In-Depth Information
Fig. 5
Representative images of the compound-treated assay cell line using the high-throughput format
(ABCDE) of the U2nesRELOC system: (
a
) vehicle control (1 % DMSO), (
b
) positive control (4 nM Leptomycin B
[LMB]), (
c
) crude extract, (
d
) active fraction from high-performance liquid chromatography (HPLC), (
e
) sample
that was eliminated by the cytox measure, (
f
)
bar graphs
that represent the quantifi cation of the effect on the
nuclear export of the reporter protein, and (
g
,
h
)
panels
demonstrating the segmentation. (
i
)
Graphs
represent
an IC50 determination curve for a putative compound. Activity is relative to cells treated with 4 nM LMB
(100 %) and carrier (DMSO, 0 %)
3.4.2 Confi rmation
of Compound Activity
by Confocal Microscopy
1. U2OS cells were cultured as described above and exposed for
1 h to equal volumes of pure compound (2
L).
2. Obtain cell cultures and rinse the cell culture media away with
several quick washes of PBS at 37 °C.
3. The cells were 100 % methanol fi xed (100
ʼ
ʼ
L/ well, 5 min) and
then incubated in 100
L/ well 1 % BSA/10 % normal goat
serum/0.3 M glycine in 0.1 % PBS Tween for 1 h to permeabi-
lize the cells and block nonspecifi c protein-protein interactions.
ʼ
4. Rinse cells in two changes of PBS with 1 % BSA for 10 min per
change.
