Staining of mitochondrial membranes with 10-nonyl acridine orange, MitoFluor Green, and MitoTracker Green is affected by mitochondrial membrane potential altering drugs.

Background: We set out to develop an assay for the simultaneous analysis of mitochondrial membrane potential and mass using the probes 10-nonyl acridine orange (NAO), MitoFluor Green (MFG), and MitoTracker Green (MTG) in HL60 cells. However, in experiments in which NAO and MFG were combined with orange emitting mitochondrial membrane potential (DeltaPsi(m)) probes, we found clear responses to DeltaPsi(m) altering drugs for both probes.

Methods: The three probes were titrated to determine whether saturation played a role in the response to drugs.

Simultaneous analysis of relative DNA and glutathione content in viable cells by phase-resolved flow cytometry.

Background: Analysis of the DNA cell cycle and glutathione content cannot be performed on viable cells, because the fluorescence emissions of the DNA-specific probe Hoechst 33342 and the glutathione-specific probe monobromobimane overlap completely. We decided to explore whether the emissions could be resolved by the singlet excited state lifetimes of the probes.

Methods: Viable cells were first incubated with Hoechst 33342 at 37 degrees C for 30 min and then with monobromobimane at room temperature for 10 min.

Enhanced immunofluorescence measurement resolution of surface antigens on highly autofluorescent, glutaraldehyde-fixed cells analyzed by phase-sensitive flow cytometry.

Background: The primary source of interference in immunofluorescence measurements by flow cytometry is background autofluorescence.

Methods: Using human lung fibroblasts (HLFs) as an autofluorescent cell model, unfixed HLFs and HLFs fixed in methanol, ethanol, formaldehyde, paraformaldehyde and glutaraldehyde were analyzed by phase-sensitive flow cytometry to compare their fluorescence intensity and lifetime histograms. Based on these results, a surface antigen on HLFs was labeled with a fluorescein isothiocyanate (FITC) conjugated antibody and fixed in glutaraldehyde, and the cells were analyzed by conventional and phase-resolved methods.

Peptides, antibodies, and FRET on beads in flow cytometry: A model system using fluoresceinated and biotinylated beta-endorphin.

Background: Particulate surfaces such as beads are routinely used as platforms for molecular assembly for fundamental and practical applications in flow cytometry. Molecular assembly is transduced as the direct analysis of fluorescence, or as a result of fluorescence resonance energy transfer. Binding of fluorescent ligands to beads sometimes alters their emission yield relative to the unbound ligands.

Flow cytometric characterization and classification of multiple dual-color fluorescent microspheres using fluorescence lifetime.

FlowMetrix (Luminex, Austin, TX) microspheres were recently introduced as a platform for bead-based assays involving antibodies, enzymes, toxins, and nucleic acids. The procedure involves classification of the microspheres by their orange and red fluorescence and quantitation of the BODIPY-tagged biological probes by their green fluorescence. In an attempt to increase the number of fluorochromes available for the biological probes, we explored the possibility of using excited singlet state lifetime as an alternative to one of the fluorochromes.