Fluorescence-Based Cell Viability Screening Assays Using Water-Soluble Oxygen Probes

Abstract

A simple luminescence-based assay for screening the viability of mammalian cells is described, based on the monitoring of cell respiration by means of a phosphorescent water-soluble oxygen probe that responds to changes in the concentration of dissolved oxygen by changing its emission intensity and lifetime. The probe was added at low concentrations (0.3 microM to 0.5 nM) to each sample containing a culture of cells in the wells of a standard 96-well plate. Analysis of oxygen consumption was initiated by applying a layer of mineral oil on top of each sample followed by monitoring of the phosphorescent signal on a prompt or time-resolved fluorescence plate reader. Rates of oxygen uptake could be determined on the basis of kinetic changes of the phosphorescence (initial slopes) and correlated with cell numbers (10(5) to 10(7) cells/mL for FL5.12 lymphoblastic cell line), cell viability, or drug/effector action using appropriate control samples. The assay is cell noninvasive, more simple, robust, and cost-effective than existing microplate-based cell viability assays; is compatible with existing instrumentation; and allows for high-throughput analysis of cell viability.

Bactenecin 7 peptide fragment as a tool for intracellular delivery of a phosphorescent oxygen sensor

“…For PC12 cells, minimal toxicity was observed after 40 h of exposure ($ 80% viability) (Figs 3 and S3). This is similar to the other PtCP conjugates described, which have been shown to be nontoxic to mammalian cells [25]. These data indicate that the PEPP0 conjugate is effectively internalized by adherent mammalian cells under standard culturing conditions employing commonly used media, such as DMEM, McCoy 5a, RPMI1640, supplemented with 1-10% serum, and also displays minimal cytotoxicity.…”

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

“…The sensitivity to oxygen for the PtCPK and PdCPK probes was within the required range and close to that of the PtCP probe (11). For the PtCPK probe, the changes in phosphorescence intensity over the entire physiological oxygen range (0 -100% of air saturation or 0 -21 kPa) are shown in Fig.…”

“…Phosphorescent oxygen probes comprising covalent conjugates of platinum(II) coproporphyrin (PtCP), platinum(II) coproporphyrin ketone (PtCPK), and palladium(II) coproporphyrin ketone (PdCPK) with BSA were synthesized using procedures described elsewhere (11,21) and are now commercially available from Luxcel Biosciences (Cork, Ireland). Oxyphor G2 probe (PdTBP dendrimer) was obtained from Harvard Apparatus (Kent, UK).…”

“…The oxygen probes based on the PtCP dye conjugated to a polypeptide carrier developed for extracellular use in oxygen consumption assays (11,12,33) were seen to incorporate a number of features important for intracellular oxygen imaging applications. Their hydrophilic nature and relatively small molecular size facilitate more simple loading procedures than those used for the particulate probes.…”

“…The group of Wilson and coworkers (8,47) have used extracellular probes based on phosphorescent dyes palladium(II) tetracarboxyphenyl porphyrin and palladium(II) tetrabenzo porphyrin (PdTBP) in low-resolution lifetime-based imaging of tissue and tumor oxygenation. A similar principle has been applied to develop simple formats for measuring biological oxygen consumption in microtiter plates on a fluorescent plate reader using phosphorescent solid-state oxygen sensors (18,27) and water-soluble probes (11). These systems, which can measure oxygen consumption noninvasively, on a microscale and with high sample throughput, have been demonstrated with bacteria (18), isolated mitochondria (14), cell lines (12), and small organisms (26).…”