


Translocation Assay
- Cells were loaded
with MitoTracker Orange (Invitrogen Inc, Cat.No.M7510) at 100 nM for 30 min
at 37oC.
- Cells were then
either untreated or UVB-irradiated at 15 mJ/cm2.
- After 24h cells
were harvested and fixed with 0.25% paraformaldehyde (PFA) for 30 min.
- Cells were then
washed in PBS/0.01% Saponin (Sigma).
- Primary mouse
antibodies against human AIF (1 mg)
were added to cell pellets and incubated for 30 min at 4oC.
- Cells were then
washed in PBS/0.01% Saponin (Sigma.)
- Secondary FITC
conjugated goat anti-mouse (0.5 mg) were
added to cell pellets and incubated for 30 min at 4oC.
- Cells were then
incubated with Hoechst 33342 20 mg/ml
- Cells were then
analysed on a BD LSRI to detect the presence of AIF-FITC (530/30nm
filter), MitoTracker Orange (575/26nm filter), Hoechst 33342 (440/40nm
filter), FSC & SCC on log scale.
- To determine the
degree of translocation of AIF from the mitochondria to nucleus cells were
homogenised in a Hepes buffer (20 mM Hepes-KOH pH7.4, 10mM KCl, 1.5 mM Mg
Cl2, 1 mM EGTA, 1 mM EDTA.
- Cells were then
re-analysed (1 x 106 events collected) using the same
instrument settings before to detect the presence of AIF-FITC (530/30nm
filter), MitoTracker Orange (575/26nm filter), Hoechst 33342 (440/40nm
filter).