Protoporphyrin IX Quantification

Quantifications of intracellular PPIX levels were determined via spectrofluorometrical measurement. Therefore, 1x10⁶ cells were seeded in a T25 cell culture flask. Approximately 12hrs later cells were treated for 6 or 24hrs with 100 g/ml 5-ALA or media alone. After the incubation period the cells were washed, trypsinized and centrifuged at 350rcf for 5min. The supernatant was removed, cells were resuspended in 1ml PBS, transferred in 1.5ml tubes and centrifuged again. PBS was removed and cell pellets were frozen at -80°C till further usage. The frozen cell pellets were thawed on ice in 1ml of 50mM tetrabutylammonium dihydrogenphosphate in acetone/methanol (1:1 v/v). Samples were treated with ultrasonic waves for 1min. In avoidance of heating up the samples the sonification was split up in 15s intervals. Subsequently, the samples were stored for 10min in the dark and centrifuged at 11000 rcf for 5min. The supernatant was used for the spectrofluorometrical measurement [18] in a FluoroMax II (Horiba Scientific, Unterhachingen, Germany) and excited at 405nm. The spectra from 450nm to 750nm was analyzed. For each experiment the untreated condition was subtracted from the 5-ALA treated measurement.