Evaluation of the derivates of phosphorescent Pt-coproporphyrin as intracellular oxygen-sensitive probes.
Several new derivatives of the phosphorescent Pt(II)-coproporphyrin (PtCP) were evaluated with respect to the sensing of intracellular oxygen by phosphorescence quenching. Despite the more favorable molecular charge compared to PtCP, self-loading into mammalian cells was rather inefficient for all the dyes, while cell loading by facilitated transport using transfection reagents produced promising results. The PtCP-NH(2) derivative, which gave best loading efficiency and S/N ratio, was investigated in detail including the optimisation of loading conditions, studies of sub-cellular localization, cytotoxicity, oxygen sensitivity and long-term signal stability. Being spectrally similar to the macromolecular MitoXpress™ probe currently used in this application, the PtCP-NH(2) demonstrated higher loading efficiency and phosphorescent signals, suitability for several problematic cell lines and a slightly increased lifetime scale for the physiological range (0-200 μM O(2)). In physiological experiments with different cell types, mitochondrial uncouplers and inhibitors performed on a time-resolved fluorescence plate reader, this probe produced the anticipated profiles of intracellular oxygen concentration and responses to cell stimulation. Therefore, PtCP-NH(2) represents a convenient probe for the experiments and applications in which monitoring of cellular oxygen levels is required.
Fercher
Andreas
A
Biochemistry Department, Cavanagh Building, University College Cork, Cork, Ireland.
Ponomarev
Gelii V
GV
Yashunski
Dmitri
D
Papkovsky
Dmitri
D
D000818:Animals; D015374:Biosensing Techniques; D002138:Calibration; D002470:Cell Survival; D003306:Coproporphyrins / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D006801:Humans; D008163:Luminescent Measurements; D015335:Molecular Probes / Q000138:chemical synthesis / Q000737:chemistry*; D015394:Molecular Structure; D009944:Organoplatinum Compounds / Q000138:chemical synthesis / Q000737:chemistry*; D010100:Oxygen / Q000032:analysis* / Q000378:metabolism; D051381:Rats; D012680:Sensitivity and Specificity; D013997:Time Factors; D014407:Tumor Cells, Cultured
D003306:Coproporphyrins; D005456:Fluorescent Dyes; D015335:Molecular Probes; D009944:Organoplatinum Compounds; D010100:Oxygen