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 (⌬⌿ m ) probes, we found clear responses to ⌬⌿ m altering drugs for both probes. Methods: The three probes were titrated to determine whether saturation played a role in the response to drugs. The effects of a variety of ⌬⌿ m altering drugs were tested for MFG and MTG at probe concentrations of 20 nM and 200 nM and for NAO at 0.1 M and 5 M, using rhodamine 123 at 0.1 M as a reference probe. Results: Incubation of GM130, HL60, and U937 cells with 2,3-butanedione monoxime (BDM), nigericin, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), 2,4-dinitro-phenol (DNP), gramicidin, ouabain, and valinomycin resulted in increases of the fluorescence intensity for MFG or MTG with only a few exceptions. The fluorescence intensity of cells stained with 0.1 M NAO increased following incubation with BDM, nigericin, and decreased for FCCP, CCCP, DNP, gramicidin, and valinomycin. The results with 5 M NAO were similar. Conclusions: MFG, MTG, and NAO appeared poor choices for the membrane potential independent analysis of mitochondrial membrane mass. Considering the molecular structure of these probes that favor accumulation in the mitochondrial membrane because of a positive charge, our results are not surprising. Cytometry 39:203-210, 2000.

We have the studied the binding of 5- ((N-(5-(N-(6-(biotinoyl)amino)hexanoyl)amino)pentyl)thioureidyl)fluorescein (fluorescein biotin) to 6.2 µm diameter, streptavidin-coated polystyrene beads using a combination of fluorimetric and flow cytometric methods. We have determined the average number of binding sites per bead, the extent of fluorescein quenching upon binding to the bead, and the association and dissociation kinetics. We estimate the site number to be ≈1 million per bead. The binding of the fluorescein biotin ligand occurs in steps where the insertion of the biotin moiety into one receptor pocket is followed immediately by the capture of the fluorescein moiety by a neighboring binding pocket; fluorescence quenching is a consequence of this secondary binding. At high surface coverage, the dominant mechanism of quenching appears to be via the formation of nonfluorescent nearest-neighbor aggregates. At early times, the binding process is characterized by biphasic association and dissociation kinetics which are remarkably dependent on the initial concentration of the ligand. The rate constant for binding to the first receptor pocket of a streptavidin molecule is ≈(1.3 ( 0.3) × 10 7 M -1 s -1 . The rate of binding of a second biotin may be reduced due to steric interference. The early time dissociative behavior is in sharp contrast to the typical stability associated with this system. The dissociation rate constant is as high as 0.05 s -1 shortly after binding, but decreases by 3 orders of magnitude after 3 h of binding. Potential sources for the time dependence of the dissociation rate constant are discussed.

Phase-sensitive multichannel detection system for chemical and biosensor arrays and fluorescence lifetimebased imaging Rev. Sci. Instrum. 71, 522 (2000)Blue light-emitting diode demonstrated as an ultraviolet excitation source for nanosecond phase-modulation fluorescence lifetime measurements Rev.

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In flow cytometry, the coincident arrival of particles becomes a major problem when high sample rates are required. For the development of our high-speed photodamage flow cytometer (ZAPPER), it was of importance to understand the behavior of cells at flow rates of around 50,000-250,000 event/s. We developed and compared two models that describe the relation between the real cell rate and the detectable single cell rate. Both the Computer Simulation model and the Input/ Output Device model show distinct optima for the cell rate. The models were compared to measurements performed on the ZAPPER-prototype. Fits of the two models to the experimental data were excellent for cycle times of 4 and 15 p s and acceptable for a 2 p s cycle time. A third model (Mercer WB, Rev. Sci. Instr. 37:1515-1521,1966) could be fitted to the experimental data, after the proportionality constant k was adapted to the experimental data. At a yield of detectable single cells of 70%, the maximum cell rates are 180,000, 100,000, and 40,000 cells/s for cycle times of 2, 4, and 15 ps, respectively. Based on these results we can now select an optimal cell rate for analysis and sorting based on criteria such as accepted cell loss. In addition, the advantages of reducing the cycle time can now be evaluated with respect to the costs of that modification.Key terms: Cell sorting, poisson, simulation, ZAPPER Early cell sorters based on the fluid switching design (2,5) sorted a single cell in about 3 ms. Droplet sorters (3) greatly improved the sorting speed. Typically, the time needed to deflect a single cell in a single droplet is in the order of 30 ps. When three droplets per cell are deflected, a FACS I1 sorter checks 2n-1= 5 droplets for coincidence (12). This increases the time in which coincidence could occur from 30 to 150 ps, thereby reducing the maximum cell rate. Sorting two or three droplets per cell is common practice as it increases the chance that the desired cell is deflected. When high sort rates are important, sorting one droplet per cell is advisable (1). Considering that the electronics, such as amplifiers and analog to digital converters (ADCs) can handle pulses of 4 ps, droplet sorting is clearly limited by the droplet frequency. Higher frequencies require smaller nozzle orifices or increased sheath fluid pressure. Nozzles smaller than 50 pm are undesirable because of clogging.High-pressure flow cytometers, such as the Lawrence Livermore National Laboratory (LLNL) high-speed sorter (91, operate at a sheath fluid pressure of 14 atm (jet speed 50 mis), a cell rate of 22,OOOis, and a droplet frequency of 220,OOOis. At these settings one out every ten droplets is occupied by a n event. However, the viability of the cells sorted at that pressure varied. Chinese hamster ovary cells survived (83%) passage through the nozzle but a muriine bone marrow sample failed to develop any colonies upon subsequent culture.Photodamage cell selection (7,101 is a relatively new approach to cell sorting. The ZAPPER (4) is a flow cytometer equipped with ...