Biomedical Engineering Reference
In-Depth Information
Fig. 4
Use of pulse shape analysis (PulSA) to monitor effects of Brefeldin A in cell
populations. (
a
) HeLa cells were treated with 2
g/mL BFA or carrier alone
(control) in suspension for 60 min. The cells were then fi xed, permeabilized,
blocked, and stained for Golgi marker, GM130 (
green
). Histograms of PulSA val-
ues for GM130 for each condition (
n
= 3 mean ± SEM) are shown here. Statistical
analysis is done using the median pulse-width values (median ± SEM; assessed
by Student's
t
-test). (
b
) Fluorescence images are 3D reconstructions, presented
as a maximum intensity projection of cells collected by cytospin. GM130 (
green
)
and DAPI (
blue
). Bars represent 10
ʼ
ʼ
M. Figure adapted from Chia et al. [
5
]
nucleotide exchange factors which play a role in membrane traf-
fi cking and organelle maintenance [
8
]. We describe below the use
of PulSA for a high-throughput quantitative analysis of Golgi frag-
mentation in cell populations (Fig.
4
):
1. Trypsinize HeLa cells for 4 min at 37 °C.
2. Suspend cells in C-DMEM before washing cells in PBS twice.
3. Treat cells with 2
g/mL BFA/C-DMEM (stock solution of
BFA is dissolved in DMSO) or carrier alone (DMSO)/C-DMEM
for 60 min/37 °C.
4. Wash cells in PBS.
5. Fix cells in 4 % PFA/PBS for 15 min/RT and then wash
in PBS.
6. Permeabilize cells in 0.1 % Triton X-100 for 4 min/RT and
then wash in PBS. Cells are blocked in 5 % FCS/PBS for at
least 30 min/RT to reduce unspecifi c binding of antibodies.
ʼ
