Biomedical Engineering Reference
In-Depth Information
Fig. 3
Use of pulse shape analysis (PulSA) to monitor the traffi cking of FLAG-furin from the plasma membrane
to the Golgi apparatus in cell populations. Histograms of PulSA values for FLAG-furin and GM130 for each time
point (
n
= 2 mean ± SEM) are shown here. The thickness of line represents SEM of the dataset at each mean
value. Figure adapted from Chia et al. [
5
]
are blocked in 5 % FCS/PBS for at least 30 min/RT to reduce
unspecifi c binding of antibodies.
7. Stain cells with Alexa568-conjugated anti-rabbit IgG (Red) for
30 min/4 °C before washing with PBS.
8. The Golgi marker GM130 is stained with mouse monoclonal
anti-GM130 antibodies for 30 min/4 °C, followed by
Alexa488-conjugated anti-mouse IgG (green) for
30 min/4 °C. Wash with PBS once between the application of
the primary and secondary antibodies (
see
Note 8
). The Golgi
marker, GM130, is used as an internal control to test for the
pulse width of the Golgi in the cell population and also to test
for changes in the Golgi during the traffi cking assay. Other
organelle markers for the early endosomes or late endosomes
can be used as well.
9. Wash cells in PBS before resuspending in 1 % (w/v) BSA/PBS
for fl ow cytometry.
10. Results are analyzed using the method described below. Briefl y,
analysis shows similar kinetics of transport for furin from the
PM to the Golgi, as previously reported [
7
] (Fig.
3
). The pulse
width for the internal control—GM130—remained similar
throughout all the time points indicating that the change in
the pulse width of furin was due to the changes in its localiza-
tion during traffi cking.
The ability to quantitate the impact of drugs on intracellular cargo
transport and organelle structure in a cell population is highly
advantageous, and this ability is limited in microscopic approaches.
Many agents or small molecules that inhibit Golgi traffi cking often
affect Golgi morphology and cause fragmentation of the Golgi.
For example, Brefeldin A (BFA) is an inhibitor of Arf guanine
3.1.2 Analysis of Golgi
Disruption After Brefeldin
A Treatment Using PulSA
