Protoporphyrin IX Quantification in Cells

Cells from 100 ml cultures were pelleted by centrifugation. The pellet was subjected to a rapid wash with pure ice-cold water and centrifuged. After the process was repeated, the pellet was incubated with 4 ml of a 9:1 mixture of acetone-0.1 N NH₄OH for 1 h at 4 °C. Cell debris was removed by centrifugation at 13000 g and 4 °C for 15 min, and the supernatant was analyzed for PPIX by mass spectrometry. The ultraviolet-visible spectrograms were plotted as previously described [4 (link), 30 (link)]. Electrospray ionization tandem mass spectrometry (ESI-MS/MS) analyses were conducted by using Finnigan LCQ Advantage Max Mass Spectrometer (Thermo Finnigan).