Figure 2

Hemin is essential for growth of Leishmania and IFP fluorescence. IFP-His expressing cell line was grown in BHI medium (BHI), YE medium supplemented with hemin (YE + hemin) or YE medium lacking hemin (YE - hemin). An ANAC42-His expressing cell line grown in BHI medium was used as a negative control. Hundred μL of the cultures were transferred to wells of a 96-well ELISA plate and supplemented with 25 μM and 250 μM of biliverdin (*; expensive) or biliverdin hydrochloride (lower price; stock solutions 25 mM in DMSO). DMSO alone was used as negative control. After addition of biliverdin or biliverdin hydrochloride, respectively, samples were incubated at 26°C. Samples were IR-scanned at 700 nm before and 30 min after addition of biliverdin or biliverdin hydrochloride using the Odyssey Infrared Imaging System. Moderate IFP fluorescence is seen in red, whereas intense IFP signal appears as white pixels in the image. Note the very low IFP fluorescence in IFP-His cultures lacking hemin (0 min incubation), and the complete absence of IFP signal in cells expressing negative control protein ANAC42-His.