Translocation Assay

 

  1. Cells were loaded with MitoTracker Orange (Invitrogen Inc, Cat.No.M7510) at 100 nM for 30 min at 37oC.
  2. Cells were then either untreated or UVB-irradiated at 15 mJ/cm2.
  3. After 24h cells were harvested and fixed with 0.25% paraformaldehyde (PFA) for 30 min.
  4. Cells were then washed in PBS/0.01% Saponin (Sigma).
  5. Primary mouse antibodies against human AIF (1 mg) were added to cell pellets and incubated for 30 min at 4oC.
  6. Cells were then washed in PBS/0.01% Saponin (Sigma.)
  7. Secondary FITC conjugated goat anti-mouse (0.5 mg) were added to cell pellets and incubated for 30 min at 4oC.
  8. Cells were then incubated with Hoechst 33342 20 mg/ml
  9. 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.
  10. 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.
  11. 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).