Imaging of cellular oxygen and analysis of metabolic responses of mammalian cells.

Many parameters reflecting mitochondrial function and metabolic status of the cell, including the mitochondrial membrane potential, reactive oxygen species, ATP, NADH, ion gradients, and ion fluxes (Ca(2+), H(+)), are amenable for analysis by live cell imaging and are widely used in many labs. However, one key metabolite - cellular oxygen - is currently not analyzed routinely. Here we present several imaging techniques that use the phosphorescent oxygen-sensitive probes loaded intracellularly and which allow real-time monitoring of O(2) in live respiring cells and metabolic responses to cell stimulation.

In vitro analysis of cell metabolism using a long-decay pH-sensitive lanthanide probe and extracellular acidification assay.

Metabolic perturbations play a critical role in a variety of disease states and toxicities. Therefore, knowledge of the interplay between the two main cellular ATP generating pathways, glycolysis and oxidative phosphorylation, is particularly informative when examining such perturbations. Here we describe a new fluorescence-based screening assay for the assessment of glycolytic flux and demonstrate the value of such analysis in assessing the cellular "energy budget.

Analysis of intracellular oxygen and metabolic responses of mammalian cells by time-resolved fluorometry.

A simple, minimally invasive methodology for the analysis of intracellular oxygen in populations of live mammalian cells is described. Loading of the cells with the phosphorescent O(2)-sensing probe, MitoXpress, is achieved by passive liposomal transfer or facilitated endocytosis, followed by monitoring in standard microwell plates on a time-resolved fluorescent reader. Phosphorescence lifetime measurements provide accurate, real-time, quantitative assessment of average oxygen levels in resting cells and their alterations in response to stimulation.

Homogeneous time-resolved fluorescence assays for the detection of activity and inhibition of phosphatase enzymes employing phosphorescently labeled peptide substrates.

A rapid, homogenous, antibody-free assay for phosphatase enzymes was developed using the phosphorescent platinum (II)-coproporphyrin label (PtCP) and time-resolved fluorescent detection. An internally quenched decameric peptide substrate containing a phospho-tyrosine residue, labeled with PtCP-maleimide and dabcyl-NHS at its termini was designed. Phosphatase catalysed dephosphorylation of the substrate resulted in a minor increase in PtCP signal, while subsequent cleavage by chymotrypsin at the dephosphorylated Tyr-Leu site provided a 3.

Quantitative detection of cell surface protein expression by time-resolved fluorimetry.

A method is introduced for quantitative detection of cell surface protein expression. The method is based on immunocytochemistry, the use of long decay time europium(III) chelate and platinum(II) porphyrin labels, and detection of photoluminescence emission from adhered cells by time-resolved fluorimetry. After immunocytochemistry, the assay wells are evaporated to dryness and measured in the dry state.

Sensing intracellular oxygen using near-infrared phosphorescent probes and live-cell fluorescence imaging.

The development and application of a methodology for measurement of oxygen within single mammalian cells are presented, which employ novel macromolecular near infrared (NIR) oxygen probes based on new metalloporphyrin dyes. The probes, which display optimal spectral characteristics and sensitivity to oxygen, excellent photostability, and low cytotoxicity and phototoxicity, are loaded into cells by simple transfection procedures and subsequently analyzed by high-resolution fluorescence microscopy. The methodology is demonstrated by sensing intracellular oxygen in different mammalian cell lines, including A549, Jurkat, and HeLa, and monitoring rapid and transient changes in response to mitochondrial uncoupling by valinomycin and inhibition by antimycin A.

Fluorescence based oxygen uptake analysis in the study of metabolic responses to apoptosis induction.

Mitochondrial activity has been shown to be centrally involved in the progression of apoptosis. The electron transport chain is a major player in this process and oxygen uptake analysis provides detailed information on its activity. Here we examined the ability of a fluorescence based oxygen uptake assay to inform on cellular responses to apoptosis induction.

Emerging applications of phosphorescent metalloporphyrins.

The subject of phosphorescent metalloporphyrins is reviewed, focusing mainly on the development and application of Pt- and Pd-porphyrins. A summary of their general chemical and photophysical properties, and guidelines for rational design of the phosphorescent labels, bioconjugates and probes is given. Examples of different detection formats and particular bio-analytical applications developed in recent years are presented.

Homogeneous assays for cellular proteases employing the platinum(II)-coproporphyrin label and time-resolved phosphorescence.

Phosphorescent platinum(II) coproporphyrin label (PtCP) is evaluated for the detection of cellular proteases by time-resolved fluorescence in homogeneous format. An octameric peptide containing the recognition motif for the caspase-3 enzyme was dual labeled with a new maleimide derivative of PtCP and with the dark quencher dabcyl. Following photophysical characterization, the quenched substrate was employed in cleavage assays for caspase-3 using Jurkat and HL60 cell lines treated with proapoptotic stimuli performed on a commercial plate reader.

Performance evaluation of the phosphorescent porphyrin label: solid-phase immunoassay of alpha-fetoprotein.

Phosphorescent conjugates of antibodies, neutravidin, and biotin (pentylamine derivative) were synthesized using previously described monofunctional labeling reagent of platinum(II) coproporphyrin-I with isothiocyanate reactive group (PtCP-NCS). These conjugates, which can be considered as standard reagents for a range of bioanalytical applications, were evaluated in solid-phase immunoassay schemes with the clinical analyte a-fetoprotein (AFP). A custom-designed time-resolved phosphorescence plate reader based on a compact and low-cost 532-nm laser and optimized for measurement of porphyrin labels was used.