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. The effects of a variety of DeltaPsi(m) altering drugs were tested for MFG and MTG at probe concentrations of 20 nM and 200 nM and for NAO at 0.1 microM and 5 microM, using rhodamine 123 at 0.1 microM 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-dinitrophenol (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 microM NAO increased following incubation with BDM, nigericin, and decreased for FCCP, CCCP, DNP, gramicidin, and valinomycin. The results with 5 microM 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. Published 2000 Wiley-Liss, Inc.

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. Samples were excited with a sinusoidally modulated laser beam (10 MHz) in a flow cytometer. The Hoechst 33342 and monobromobimane lifetimes and fluorescence intensities were resolved by using phase-sensitive detectors. RESULTS: The observed singlet excited state lifetimes were 1.5 ns for Hoechst 33342 and 12 ns for monobromobimane. The glutathione (GSH) content was shown to increase as cells (GM130, HL60, U937) progressed through the cell cycle. However, after the data were corrected for differences in cell volume, it was found that the GSH concentration was constant throughout the cell cycle of the exponentially growing cells. CONCLUSIONS: Phase-resolved flow cytometry provides a means for the specific analysis of the GSH content/concentration as a function of the cell's position in the DNA cell cycle in viable cells.

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. RESULTS: The lifetimes of unfixed and ethanol-, methanol-, paraformaldehyde- and formaldehyde-fixed HLFs were in the 1.7-1.9 nanosecond (ns) range, with coefficients of variation 25-35%. Since the autofluorescence lifetime histograms of unfixed and fixed HLFs partially overlapped the 3.5 ns lifetime histogram of FITC-labeled microspheres, which were used to approximate FITC-antibody labeling of HLFs, the ability to resolve FITC-labeled probe, based on differences in the FITC and autofluorescence lifetimes, was severely limited. When HLFs labeled with an FITC-antibody cell-surface marker were fixed in glutaraldehyde (autofluorescence lifetime 0.9-1.4 ns, coefficient of variation approximately 11%) and analyzed by phase-resolved methods, the results showed that FITC-antibody labeling could be readily resolved from background autofluorescence. CONCLUSIONS: Phase-sensitive detection improves the immunofluorescence measurement resolution of surface antigens on highly autofluorescent, glutaraldehyde-fixed cells. Cytometry 37: 275-283, 1999. Published 1999 Wiley-Liss, Inc.

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. For a set of 20 dual-color microspheres the excited singlet state lifetimes were measured using the total emissions (>515 nm), the orange emissions (515-600 nm), and the red emissions (>665 nm). The microspheres could not all be resolved in bivariates of fluorescence intensity versus excited singlet state lifetime. However, 13 of the microspheres could be resolved using the total emissions and lifetime. Although this result required both fluorochromes, the merits and limitations of this approach to other systems are briefly discussed.

Evaluation of the silicon phthalocyanine pc 4 for photodynamic bone marrow purging.

The silicon phthalocyanine Pc 4 was tested as a photosensitizer for the selective photoinactivation of malignant cells in bone marrow transplantation samples. Using a murine model system, incubation of 1.5×107 cells/mL with 15 nM Pc 4 followed by exposure to red light (λ>600 nm, fluence of 18 J/cm2) was shown to result in a greater than 6 log10 reduction of the clonogenic growth for the murine cell lines ABE-8.1/2, BC3A and L1210. The clonogenic growth of WEHI-3 and P815 cells was reduced by more than 5 log10 and more than 3 log10, respectively. Late murine hematopoietic progenitor cells were less sensitive than cancer cells; the surviving fractions were 0.084 for the colony forming unit, megakaryocyte (CFU-Mk); 0.038 for the colony forming unit, granulocyte macrophage (CFU-GM); 0.0018 for the colony forming unit, mix (CFU-mix) and <0.003 for burst forming units, erythroid (BFU-E). Early hematopoietic progenitor cells, assayed by the in vitro cobble stone area forming cell assay, were not affected by the photodynamic treatment. Likewise, in vivo assays of early hematopoietic progenitor cells showed no reduction of their ability to repopulate the bone marrow. Irradiation of the samples following incubation of 1.5×106 cells/mL with Pc 4 resulted in increased photosensitivity of all cell types, including the early and late hematopoietic progenitor cells. Flow cytometric analysis of Pc 4 uptake by the cells revealed that the increased photosensitivity could be traced to increased Pc 4 uptake; however, Pc 4 uptake among cell types did not correlate with photosensitivity. When mixed with bone marrow (BM) cells, Pc 4 uptake in the cell lines increased as the fraction of BM increased from 0.5 to 0.95. These observations suggest that Pc 4 may be a suitable photosensitizer for bone marrow purging. © 1998 Society of Photo-Optical Instrumentation Engineers.

Homing of fluorescently labeled murine hematopoietic stem cells.

PKH-26 was used as a viable fluorescent membrane stain for murine hematopoietic stem cells. The presence of the dye on the cells was shown not to interfere with their ability to form day-8 and -12 spleen colonies in lethally irradiated mice. To study their in vivo homing behavior in detail, 10(4) labeled cells from a population enriched for CFU-S were injected intravenously into nonirradiated mice and into mice irradiated 3 hours previously. At 17, 41, and 65 hours after injection, the numbers of labeled cells per organ were quantified using the specialty developed flow cytometric fluorescence hypercompensation procedure for the detection of rare events, which allows a detection sensitivity of 1 per 10(6). Spleen homing in irradiated and nonirradiated mice was virtually identical, whereas homing to nonirradiated bone marrow was 2.5 times higher than to irradiated bone marrow. This indicates a different homing mechanism for spleen and bone marrow. The results of this direct homing assay were placed in perspective with results of indirect homing studies from the literature, introducing a new "h-factor." From the CFU-S data, putative specific enrichment factors for spleen-specific and bone marrow-specific homing were derived. Examination of the fluorescence intensity distribution among the labeled cell population indicated that virtually all cells started to proliferate rapidly after injection into both irradiated and nonirradiated animals. This indicates that specific signals from stromal elements in the stem cell niches are needed to keep the cells quiescent and that the majority of the transplanted stem cells do not home to such niches. The potential use of PKH-26 for in vivo characterization of stem cell niches is discussed.

High-speed photodamage cell selection using a frequency-doubled argon ion laser.

A flow cytometer was developed for the high-speed "sorting" of desired cells by selectively irradiating (zapping) the undesired cells from a population. After previous efforts to photoinactivate cells with photosensitizers had failed, it was decided to exploit the photosensitivity of the cell's DNA at 257 nm. It was shown that a 257 nm laser output power of 20-100 mW was sufficient to induce a 4.5 log cell kill after the cells were processed through a focused 257 nm laser beam. Experiments proved that the photodamage flow cytometer (ZAPPER) could selectively photoinactivate cells at rates over 22,000 events/s, and selection purities ranged from 81% to 100%. The yields of the desired cells depended on the selection mode. In the Enrichment mode, the zap laser was not aimed at the jet, and only undesired cells were exposed to a brief ultraviolet (UV) pulse after modulation of the UV laser beam. The yields of desired cells ranged from 95% to 105%. In the Purge mode, the zap laser beam was aimed onto the jet, and only desired cells were allowed to pass after deflection of the UV laser beam; the yields of desired cells ranged from 12% to 52%. The cause of the reduced yields in the PURGE mode was traced to the fact that the Electro-Optic Modulator was used to modulate the zap laser proved too slow for the intended application. The lifetime of the frequency-doubling crystal used for the generation of the 257 nm beam was found to be limited to several days.(ABSTRACT TRUNCATED AT 250 WORDS)

Classmode: a new data format for real-time multiparameter data analysis and data compression.

A new data acquisition and analysis format (classmode) was developed that allows real-time data classification in a flow cytometer. In our cytometer, detected events were classified in real time by their presence or absence in a set of look-up tables (LUT). A modification of the cytometer hardware allows the exclusive transfer of the LUT data to the acquisition/storage computer. Using a combination of 8 LUTs, the analyzed events can be classified into 256 subpopulations. Real-time data classification results in an increased data transfer rate and a significant compression of the data.

Avidin-EITC: an alternative to avidin-FITC in confocal scanning laser microscopy.

Detection of fluorescein-5-isothiocyanate (FITC)-labeled conjugates is suboptimal in two-color confocal scanning laser microscopy (CLSM). This limits the detection of small, dimly fluorescent targets. We explored the possible advantages of applying eosin-5-isothiocyanate (EITC) conjugated to avidin (Av-EITC) as an alternative for Av-FITC in CSLM. Despite the lower quantum efficiency of EITC, we found that the measured Av-EITC and Av-FITC emission intensities were similar as a result of the standard filter combinations used for simultaneous two-color detection in the Bio-Rad MRC 600 CSLM. The advantage of Av-EITC was that its fading characteristics compared very favorably to those of Av-FITC. An excitation intensity-dependent increase in Av-EITC fluorescence was observed, followed by an exponential decrease. This increase in fluorescence allows longer observation times, averaging of several scans without loss of brightness, and thus detection of dimly fluorescent targets by CSLM.

Coincidence in high-speed flow cytometry: models and measurements.

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 microseconds and acceptable for a 2 microseconds 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 microseconds, 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.

INCA: software for consort 30 analysis of flow cytometric calcium determinations.

The INCA program converts Consort 30-generated fluorescence list mode data collected from Indo-1-stained cells to absolute intracellular calcium concentrations (nM Ca2+i). The calcium data are plotted vs. time, allowing the user to analyze the fractions of cells responding to a given stimulus. Converted files can be restored to disk after replacing FL1 and FL2 with time and calcium, respectively, for future analysis.

Interaction of hyperthermia and radiation in tolerant and nontolerant HeLa S3 cells: role of DNA polymerase inactivation.

The activities of DNA polymerase alpha and beta were measured in tolerant and nontolerant HeLa S3 suspension cells. The heat-inactivation of the enzymes and their recovery when cells were incubated at 37 degrees C after the heat challenge was compared to the synergistic action of heat and radiation and its disappearance at the level of cell survival. Thermotolerant cells were radiosensitized by heat similarly to nontolerant cells, but the sensitization decreased more rapidly in the tolerant cells when time at 37 degrees C was allowed between the two treatments. For polymerase activities the extent of inactivation, as well as the kinetics of recovery, were similar in tolerant and nontolerant cells. The results show that the activities of DNA polymerase alpha and beta do not always correlate with the extent of heat radiosensitization. It is concluded that heat inactivation of these enzymes may not be taken as a general cause for the synergistic effect of hyperthermia and radiation. As an alternative mechanism, changes in nuclear protein binding due to cellular heating are suggested, since these correlate well with effects observed for radiosensitization under different experimental conditions, including the use of thermotolerant cells.

The use of DNA flow cytometry in the diagnosis of triploidy in human abortions.

The ploidy of placental tissue from 59 abortions was determined using DNA flow cytometry. The specimens were also screened histologically for features considered to be characteristic of a partial mole. Triploidy was found in six cases; three of these cases presented histologically as partial moles. From the four specimens histologically classified as partial moles, three appeared to be triploid. The most specific histologic features of triploidy were cystic change of the placental villi and trophoblastic hyperplasia with vacuolation of the syncytiotrophoblast; however, these changes were also found in some of the nontriploid abortions. Irregularity of villous contours was less specific.

Detection and isolation of antigen-specific B cells by the fluorescence activated cell sorter (FACS).

A method is described for the isolation of antigen-specific B cells from immunized and subsequently boosted mice. Antigen-specific B cells were stained by incubation with fluorescein isothiocyanate (FITC)-labelled antigen and then detected and isolated in a fluorescence activated cell sorter (FACS). Ovalbumin (OVA) and Helix pomatia haemocyanin (HPH) were used as antigens in this procedure, yielding relative amounts of antigen-FITC-binding lymphocytes of 0.9 +/- 0.4% and 3.5 +/- 3.1%. The FITC-positive cells were visible as distinct cell populations in the FACS-generated histograms. All antigen-FITC-binding cells were B cells, as shown by double staining with phycoerythrin-conjugated anti-mouse Ig In addition, as tested in a spot-ELISA, the sorted, antigen-FITC-binding cell population contained almost the entire population of antigen-specific antibody-producing B cells. However, sorting had a negative influence on the antibody production capability of the sorted cells. Through washing of isolated spleen cells in the procedure before labelling with antigen-FITC proved to be essential for the specific detection of antigen-specific B cells, since staining without prior washing resulted in antigen-FITC binding to all B cells. This 'nonspecific' staining phenomenon was caused by the presence of antibodies, specific for the immunizing/boosting antigen, which were also present in the spleen cell suspension. These antibodies formed immune complexes with antigen-FITC and bound to Fc receptors present on all B cells, interfering in this way with any specific binding of antigen-FITC to sIg on the B cells.

Comparison of helium-neon and dye lasers for the excitation of allophycocyanin.

Allophycocyanin (APC) has a broad absorption spectrum permitting several different lasers to be used to excite this dye in a flow cytometer. A comparison was made between a dye laser and a helium-neon (HeNe) laser for the excitation of APC as an immunofluorescent chromophore. The ratio of fluorescence of stained to unstained lymphocytes (signal to background) was used to assess differences in sensitivity. In determining the best wavelength for operating the dye laser, it was found that there was little difference in the ability to separate the positive-labelled cells from the unstained cells using 600 nm or 633 nm light for excitation of APC. A study of the effect of laser power on the signal to background identified a nonlinear relationship. It was found that the sensitivity obtained with 47 mW of 633 nm light from a HeNe laser was near the maximum attainable. This sensitivity was comparable to that obtained using phycoerythrin as an immunofluorescence chromophore. APC had the added advantage of being applicable to the study of highly autofluorescent cells. Exciting this chromophore using red light dramatically decreased the autofluorescence observed even on alveolar macrophages.