Macrophage activation by synthetic peptides. II. The effect of oxidative burst products on the fluorescence kinetics of macrophages stained with fluorescein diacetate

Our previous study has shown that the intensity of macrophages in a medium containing fluorescein diacetate (FDA) retains a certain constant level ready to fall upon the addition of stimulants. The decrease in fluorescence was attempted to explain by secretion of oxygen metabolites during the activation process. The decrease in fluorescence is much smaller in the medium containing catalase and superoxide dismutase. Hydrogen peroxide (50 nM and higher) causes a reversible decrease in the fluorescent intensity of resting macrophages. These experiments suggest that the oxidative burst products may serve as regulators of the feedback control during macrophage activation.     

Macrophage activation by synthetic peptides. I. Kinetic changes in the transport of fluorescein anions across the plasma membrane of macrophages

As shown by cytofluorimetric technique, fluorescein anions formed in macrophages due to hydrolysis of fluorescein diacetate (FDA) release into the extracellular medium through the probenecid-inhibitable transport system of organic acids. Technical procedures have been elaborated to record separately the process of FDA hydrolysis characterising the activity of intracellular esterases, and the fluorescein anion transport representing secretion of organic acids by macrophages. It has been established that the tetrapeptide tuftsin stimulates the cell esterase activity without affecting the rate of fluorescein efflux. The peptide KPR (Lys-Pro-Arg) decreases both the esterase activity and the fluorescein anion efflux.     

Potential problems with fluorescein diacetate assays of cell viability when testing natural products for antimicrobial activity.

There are two potential problems in the use of fluorescein diacetate (FDA) as a measure of cell viability. The first is the hydrolysis of FDA to fluorescein in the absence of live cells and the second is the quenching of fluorescence by assay solutions. We show that common media components such as tryptone, peptone and yeast extract all promote hydrolysis of FDA in the absence of live cells, as do Tris-HCl and sodium phosphate buffers. As a consequence, various microbiological media promote hydrolysis of FDA in the absence of live cells. Different media were also shown to reduce the amount of visible fluorescence of fluorescein. Diluting the medium decreases the background hydrolysis of FDA as well as increases the amount of visible fluorescence. Both problems should be considered when using FDA as an indicator of cell viability when testing natural products for antimicrobial activity.     

Rapid analytical technique for the assessment of cell metabolic activity in marine microalgae

A standard method for the assessment of cell viability has been developed for marine phytoplankton using an inexpensive stain, fluorescein diacetate (FDA), at .75 microM for 10 min. A flow cytometer was used as the fluorescence detector, providing an assessment of viability for each individual particle. Cell size and chlorophyll fluorescence per cell were assessed simultaneously, permitting an assignment of viability to specific subpopulations, thus increasing the power of the technique. A reasonable correspondence between FDA mean fluorescence intensity per cell and an independent metabolic indicator, photosynthetic capacity measured by 14C, was found. Both FDA mean fluorescence intensity and photosynthetic capacity vary as a function of cell volume. Recovery after extended periods of darkness indicate that cells that are FDA negative may not be dead, but merely quiescent or inactive.     

Changes in the permeability of amphibian embryo envelope for fluorochromes under the action of ultrasound

It was determined whether high-frequency ultra sound can change the permeability of gray toad Bufo bufo and grass frog Rana temporaria under the action of high-frequency ultrasound. The changes in the permeability of embryonic envelope were assessed by using slowly penetrating fluorochromes ANS, FDA, and fluorescein. It was found that the ultrasound of 0.88 MHz and 0.4 - 0.7 W/cm2 intensity increased the permeability of amphibian embryonic envelope for ANS and FDA, whereas the ultrasound of 2.64 MHz and the same intensity increased that for fluorescein with the retention of low permeability for FDA. Embryos continued the normal development after treatment with ultrasound under these conditions.     

Staining with Fluorescein Diacetate Correlates with Hepatocyte Function

To establish the importance of fluorescein diacetate (FDA) as a viability stain for cultured hepatocytes. we hypothesized that FDA staining would correlate positively with hepatocyte viability and function. Mixtures of live and dead cells were stained with FDA and scanned by flow cytometry. A close correlation was observed between the live cell fraction and percent viability as determined by FDA staining (R² = 0.962). Hepatocytes were also sorted into low fluorescence and high fluorescence groups. Both albumin production and lidocaine metabolism (P-450 activity) were significantly increased in the high fluorescence group compared to the low fluorescence group. An automated, fluorescence-activated assay was useful for rapid assessment of hepatocyte viability. In addition. the intensity of green fluorescence following staining with FDA correlated well with two specific measures of hepatocyte function.     

Fluorescence properties of free and protein bound fluorescein dyes. I. Macrospectrofluorometric measurements.

Excitation and emission properties of fluorescein derivatives were studied macrofluorometrically. Measurements were performed with solutions of various concentrations (0.07-100 microgram/ml) of free sodium fluorescein prepared from fluorescein diacetate (FDA), fluorescein isothiocyanate (FITC) and FITC bound to rabbit gamma-globulin. Both excitation and emission spectra as well as fluorescence intensities at constant excitation/emission wavelengths (496/515 nm) were recorded. The findings indicate that (1) FDA gives about twice the fluorescence intensity compared to equal concentrations of FITC. (2) The fluorescence properties of FITC upon excitation with blue light (lambda = 496 nm) are only slightly altered by the conjugation to rabbit gamma-globulin. (3) Considerable quenching due to conjugation could, however, be shown to occur upon UV excitation (lambda = 340 nm). (4) Fluorescence emission excited by visible blue light (496 nm) increases linearly to dye concentration in a range of 0.07-2.5 microgram/ml. Beginning at 5 microgram/ml (10-(5) M/1) all three compounds show a sharp decrease of fluorescence intensity with further increasing concentration. Practical aspects of these data for the immunofluorescence method are discussed.     

A Method for Producing Dust-, Streak- and Hole-Free Formvar Films in Laboratories Having High Atmospheric Humidity

To establish the importance of fluorescein diacetate (FDA) as a viability stain for cultured hepatocytes. we hypothesized that FDA staining would correlate positively with hepatocyte viability and function. Mixtures of live and dead cells were stained with FDA and scanned by flow cytometry. A close correlation was observed between the live cell fraction and percent viability as determined by FDA staining (R² = 0.962). Hepatocytes were also sorted into low fluorescence and high fluorescence groups. Both albumin production and lidocaine metabolism (P-450 activity) were significantly increased in the high fluorescence group compared to the low fluorescence group. An automated, fluorescence-activated assay was useful for rapid assessment of hepatocyte viability. In addition. the intensity of green fluorescence following staining with FDA correlated well with two specific measures of hepatocyte function.     

Applicability of the fluorescein diacetate assay for metabolic activity measurement of Microcystis aeruginosa (Chroococcales,Cyanobacteria)

The fluorescein diacetate (FDA) assay has been widely used to measure metabolic activity in phytoplankton. It was found that FDA fluorescence values did not decrease in some stressed cells, demonstrating that the applicability of the method needs to be assessed further in the context of growth‐influencing conditions. In the present study, changes of FDA fluorescence values were studied in bloom‐forming cyanobacterial Microcystis aeruginosa Kütz cells under stress conditions such as nitrogen (N) or phosphorus (P) deficiency, or darkness and low temperature (10°C), respectively. The results demonstrated that esterase activity decreased immediately in dark‐stressed cells, which correlated with the decline of biomass and photosynthetic activity. Under the other three stress conditions, however, especially at low temperature, the cells lost photosynthetic activity but had the highest esterase activity, which was five times higher than the control group. These findings contrast with the assay criteria that the expression of a stain should reflect the change of photosynthetic activity and that stressed cells should have a lower staining intensity than the control cells. According to these results, the esterase activity response was dependent on environmental factors. Furthermore, higher fluorescence intensity did not mean higher metabolic activity, but a discrepant value indicated a severe stress.     

Fluorescein diacetate used as a vital stain for labeling living pollen tubes

Snapdragon and tobacco pollen treated with fluorescein diacetate (FDA, 10 μg/ml) for 40 min could germinate and grew well in FDA-free medium. The pollen tubes showed bright fluorescence of the protoplasm when they were living. Thus the FDA method can be used, in addition to its previous usage for viability test of cells, also for vital staining of pollen tubes, and may be valuable to the study of other living cells as well.     

Analysis of enzyme kinetics in individual living cells utilizing fluorescence intensity and polarization measurements

BACKGROUND: The Cellscan mark-S (CS-S) scanning cytometer was used for tracing enzymatic reactions in the same individual cells under various physiological conditions over periods of minutes. On-line reagent addition and changes in the experimental conditions (buffers, ions, substrates and inhibitors) were performed. METHODS: Kinetic events were monitored by fluorescence intensity (FI) and fluorescence polarization (FP) measurements of fluorescein diacetate (FDA) and chloromethyl fluorescein diacetate (CMFDA) intracellular hydrolysis. FP measurements have been used to assess the intracellular marker's mobility restrictions. RESULTS: Kinetic measurement along 1000 s of FDA labeled individual Jurkat T cells, indicated variation of 65% for FI(t) and approximately 10% for FP(t). While FI increased linearly with time, FP(t) decreased nonlinearly and asymptotically, reaching a constant value. The FP(t) of CMFDA-labeled cells was different from that of FDA-labeled cells. Average cellular Km of 3.9 microM was calculated from individual cell FDA hydrolysis curves. CONCLUSIONS: (1) Analysis of the reaction kinetics of intracellular enzymes can be refined by using FP measurements of the products of fluorogenic substrates in addition to the FI measurements. (2) Subpopulations or individual cells could be classified according to their reaction rates. (3) A specific dependence of FP(t) on type of enzyme substrate is suggested.     

Flow cytometric measurement of pollutant stresses on algal cells

The lichen Usnea fulvoreagens (R?s). R?s. was treated with four pH levels (5.5, 4.5, 3.5, and 2.5) of simulated acid rain (sulfuric acid, nitric acid, and a 1:1 combination of both) and automobile exhaust. The samples were dissociated and analyzed by a Becton-Dickinson FACS 440 flow cytometer. Analyses included measurement of chlorophyll autofluorescence and fluorescence due to uptake of fluorescein diacetate (FDA) and calcofluor white M2R (CFW). Cell parameters measured were esterase activity (FDA), membrane permeability (FDA, CFW), and intracellular pH (FDA). Mean fluorescence intensity from FDA staining and numbers of events were incorporated with autofluorescence information to produce a "stress index" of relative cell stress. Results indicated that highly stressed samples (lower pH treatments and greater exposure to exhaust) exhibited a low "stress index" of FDA fluorescence.     

Fluorometric test of cell membrane integrity

A fluorometric test for assessing cell membrane integrity of bone marrow, based on the use of fluorescein diacetate (FDA), is described. It is demonstrated that the amount of fluorescein extracted from the labeled cells is directly proportional to the number of intact cells and that this relationship is not affected by the presence of the dead cells. The experimental conditions required for the accuracy of this test are as follows: (1) During the incubation with FDA the number of cells should not exceed ca. 3 × 10⁶ cells/ml. (2) With the fluorescein concentration of 2 μg/ml, the incubation temperature should be within the range of 20 to 30 °C for a period of 10 to 20 min. (3) To prevent the loss of fluorescein by labeled cells during washing, the cells must be maintained at a temperature near 0 °C. (4) If the cells are contaminated by hemoglobin, an appropriate correction of the fluorescein readings must be made. Provided that the controls are appropriately adjusted, the accuracy of the test should not be affected by the presence of serum and/or of DMSO in the reaction medium. Other potential sources of error may be the enhanced loss of fluorescein due to the presence of albumin or platelet-absorbed serum and the possible activation of esterases before the FDA treatment. It is concluded that the results of this test should be interpreted in terms of relative changes of the collective cell membrane integrity rather than in terms of cell viability, and that these results may be more meaningful than those based on the visual assessment of either the FDA-labeled cells or of the cells subjected to the dye exclusion test.     

Quantitation of the binding, uptake, and degradation of fluoresceinylated neoglycoproteins by flow cytometry

The fluorescence properties of the fluorescein residues bound to a protein are used to analyze by flow cytometry the neoglycoproteins' endocytosis mediated by membrane lectins of Lewis lung carcinoma cells (3LL cells). The quantum yield of fluorescein bound to a protein is dependent on the number of fluorophore molecules bound to a protein molecule and the pH of the environmental medium. The mean fluorescence intensity of a fluorescein molecule bound to a protein decreases when the number of fluorescein residues per protein molecule increases. However, after proteolytic digestion, the mean fluorescence intensity of a fluorescein molecule is constant and equal to that of free fluorescein. The binding of fluorescein-labeled alpha-glucosylated serum albumin to 3LL cells at 4 degrees C can easily be determined by flow cytometry because under these conditions the environmental pH is neutral, and the neoglycoprotein is not degraded. When the cells are incubated at 37 degrees C in the presence of a fluorescein-labeled neoglycoprotein, the fluorescence intensity of a cell is low because of the low pH of endosomes and lysosomes but is increased upon a postincubation at 4 degrees C in the presence of monensin, a proton/sodium ionophore. The extent of the proteolytic digestion of an endocytosed neoglycoprotein can be assessed by comparing, upon a monensin postincubation at 4 degrees C, the high cell-associated fluorescence of cells incubated in the absence of leupeptin (an inhibitor of lysosomal proteases) and the relatively low fluorescence intensity of cells incubated in the presence of leupeptin.     

Complement-mediated lysis of pigeon erythrocyte ghosts analysed by flow cytometry. Evidence for the involvement of a ''threshold'' phenomenon.

Flow-cytometric analysis of complement-mediated lysis of antibody-coated pigeon erythrocyte ghosts containing fluorescein was carried out to determine whether lysis involved a gradual release of fluorescein or a 'threshold' release from individual cells. Antibody-coated ghosts were comprised of three subpopulations identified by fluorescence and scatter (size). These were: (a) highly fluorescent, medium scatter, (b) medium fluorescence, high scatter, and (c) low (or zero) fluorescence, low scatter. Lysed ghosts and isolated nuclei were identified by fluorescence microscopy and scanning electron microscopy. Fluorescence distributions analysed by flow cytometry indicated that, after complement attack, those ghosts remaining intact retained all their fluorescent label. A time course of changes in ratios of the three subpopulations indicated that once lysis of an individual ghost was initiated, release of label was complete within 1 min; no stages of intermediary fluorescence appeared, and those ghosts remaining at the end of the experiment retained the same fluorescence intensity as control ghosts. The results supported the hypothesis that complement-mediated cell lysis is a 'threshold' phenomenon; a submaximal response by a cell population representing a complete response by only some of the cells rather than a partial response by all of the cells.     

pH in the endosome. Measurements during pinocytosis and receptor-mediated endocytosis

By fluorescence spectroscopy, the average pH within endocytic compartments was determined during endocytosis of fluorescein conjugates by macrophages and hepatocytes. In mouse macrophages and hepatocytes fluorescein conjugates taken up either in the fluid phase or by binding to cell surface receptors were rapidly transferred to an acidic compartment (pH 5-5.5). The half-time for this process was generally less than 4 min. The pH within yeast-containing phagosomes was also rapidly reduced to similar levels, following a unique and transient increase. In each case, the acid endosomal compartments involved probably do not contain lysosomal enzymes. When fluorescein conjugates of asialoglycoproteins were internalised by hepatocytes at 20 degrees C, no proteolysis occurred within the acidic endosome until the temperature was raised. Fluorescein conjugates of concanavalin A (conA) and polylysine were relatively more slowly internalised by macrophages. The half-times for uptake, estimated by fluorescence change, were comparable with the turnover time for bulk plasma membrane. The relatively high average pH experienced by these conjugates indicated that a small proportion of these non-specific cell-surface labels was always in contact with the extracellular medium.     

Detection of respiratory enzyme activity in Giardia cysts and Cryptosporidium oocysts using redox dyes and immunofluorescence techniques.

The fluorescent redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), combined with fluorescein-labeled antibodies, was tested for the simultaneous detection of the respiratory electron transport system (ETS) activity and enumeration of Giardia cysts and Cryptosporidium oocysts by spectral microfluorometry and epifluorescence microscopy. The reduction of CTC and p-iodonitrotetrazolium violet (INT), a non-fluorescent redox dye, was compared with propidium iodide (PI) and fluorescein diacetate (FDA) for the measurements of Giardia cyst viability over time. According to the PI and FDA staining techniques, nearly 60% of the cysts tested viable at the beginning of the observations; after 21 days their viability decreased to 5%. The redox dyes indicated that approximately 4-10% of the cysts were metabolically active 48 h after they were shed, followed by a decline in enzyme activity to near undetectable levels after 4 days. Spectral analysis on individual cysts indicated that the fluorescence emission of the reduced CTC and the fluorescein-labeled antibodies is distinctive for each compound and suitable for their simultaneous determination by microphotometry, flow cytometry and epifluorescence microscopy. The fluorescence signal remained without alteration when the cysts were transferred onto microscope slides coated with an optical embedding medium and stored at -20 degrees C. The fluorescence intensity of the reduced CTC, when properly standardized, can provide quantitative measurements of ETS activity of the cysts. This is the first report of a method to determine enzyme redox activity on intact cysts applicable to water, laboratory and animal samples.     

Fluorescein labeling of Fab' while preserving single thiol

We determined the conditions for labeling rabbit Fab' with fluorescein isothiocyanate to provide maximal fluorescence intensity compatible with the preservation of integrity of its single thiol residue. We found that we could reproducibly substitute Fab' with two fluorescein moieties in 30 min at room temperature at pH 8.9. We achieved a high level of fluorescence intensity while we preserved the integrity of more than half of the existing thiols.     

Quantification of metabolic activity of cultured plant cells by vital staining with fluorescein diacetate

The metabolic activity of suspension cultures of Sonneratia alba cells was quantified by measurement of the hydrolysis of fluorescein diacetate (FDA). FDA is incorporated into live cells and is converted into fluorescein by cellular hydrolysis. Aliquots (0.1–0.75 g) of S. alba cells were incubated with FDA at a final concentration of 222 μg/ml suspension for 60 min. Hydrolysis was stopped, and fluorescein was extracted by the addition of acetone and quantified by measurement of absorbance at 490 nm. Fluorescein was produced linearly with time and cell weight. Cells of S. alba are halophilic and proliferated well in medium containing 50 and 100 mM NaCl. Cells grown in medium containing 100 mM NaCl showed 2- to 3-fold higher FDA hydrolysis activity than those grown in NaCl-free medium. When S. alba cells grown in medium supplemented with 50 mM NaCl were transferred to fresh medium containing 100 mM mannitol, cellular FDA hydrolysis activity was down-regulated after 4 days of culture, indicating that the moderately halophilic S. alba cells were sensitive to osmotic stress. Quantification of cellular metabolic activity via the in vivo FDA hydrolysis assay provides a simple and rapid method for the determination of cellular activity under differing culture conditions.