Flow cytometry of leukocytes after intravenous fluorescein angiography

Blood specimens were obtained for T lymphocyte analysis from patients undergoing intravenous fluorescein angiography (IVFA). Mononuclear cells were prepared by differential density centrifugation and analyzed by flow cytometry (FCM). Prior to IVFA, there was very little autofluorescence in the mononuclear cell population. After IVFA, there was an increase in the fluorescence of the mononuclear cells. Though barely visible by fluorescence microscopy, the fluorescence was uniformly distributed throughout the cytoplasm. By FCM, fluorescein was detected in 27% of the lymphocytes, 61% of the monocytes, and 75% of the granulocytes. Care must be taken when interpreting flow-cytometric lymphocyte subset data because of the increased nonspecific fluorescence of mononuclear cells in patients who have undergone IVFA.     

A single laser method for subtraction of cell autofluorescence in flow cytometry

In flow cytometry cell autofluorescence often interferes with efforts to measure low levels of bound fluorescent antibody. We have developed a way to correct for autofluorescence on a cell-by-cell basis. This results in improved estimates of real staining and better separation of the fluorescence histograms of stained and non-stained cells. Using a single laser, two-color fluorescence measurement system and two-color compensation electronics, autofluorescence and one fluorescent reagent are measured (rather than two fluorescent reagents). With fluorescein-conjugated antibodies the signal in the 515 to 555 nm range (green fluorescence) includes both fluorescein emission and part of the cellular autofluorescence. In the cases we have investigated, autofluorescence collected at wavelengths above 580 nm ("red") is well correlated with the green autofluorescence of the cells. A fraction of this red fluorescence is subtracted from the green fluorescence to produce an adjusted fluorescein output on which unstained cells have zero average signal. Use of this method facilitates the selection of rare cells transfected with surface antigen genes. Culture conditions affect the level of autofluorescence and the balance between red and green autofluorescence. When applied with fluorescein-conjugated reagents, the technique is compatible with the use of propidium iodide for live/dead cell discrimination.     

Purification and characterization of methylmalonyl-CoA mutase from a photosynthetic coccolithophorid alga, Pleurochrysis carterae

Low activity (about 4 mU/mg protein) of 5'-deoxyadenosylcobalamin-dependent methylmalonyl-CoA mutase (MCM; EC 5.4.99.2) was found in a cell homogenate of a photosynthetic coccolithophorid alga, Pleurochrysis carterae. Most of the enzyme occurred as the apo-enzyme, which was labile during purification. The holo-enzyme, which was converted from the apo-enzyme by incubation with 10 microM 5'-deoxyadenosylcobalamin at 4 degrees C in the dark, was purified to homogeneity and partially characterized. An apparent molecular mass for the enzyme of 150+/-5 kDa was calculated by Superdex 200 pg gel filtration. SDS-polyacrylamide gel electrophoresis of the purified enzyme gave a single protein band with an apparent molecular mass of 80+/-5 kDa, indicating that the P. carterae enzyme occurs as a homodimer. Some properties of methylmalonyl-CoA mutase from P. carterae were studied.     

Changes in fluorescence intensity of selected leukocyte surface markers following fixation.

BACKGROUND: Immunophenotyping of blood leukocytes often involves fixation with paraformaldehyde prior to cytometry analysis. However, the influence of cell type and marker specificity on the stability of fluorescence intensity after fixation has not been well studied. METHODS: Human whole blood was stained using a panel of fluorescein isothiocyanate-labeled antibodies to surface markers. Unfixed and fixed samples were analyzed by flow cytometry at 0, 2, 4, 6, 24, 48, and 96 h after staining. Fluorescence measurements were converted to molecules of equivalent soluble fluorochrome for comparison. RESULTS: Fixation caused a significant decrease in both forward and side scatter at 48 h which required gating adjustments to achieve resolution of cell populations. The autofluorescence increased progressively in fixed samples (ninefold at 96 h for monocytes). Variable decreases in marker-associated fluorescence became apparent after correction for autofluorescence. The magnitude of the decrease at 96 h varied with cell type and marker, from 5% for CD32 on monocytes to 39% for CD16 on neutrophils. CONCLUSION: The change in fluorescence intensity following staining and fixation of leukocytes varies with cell type and surface marker. Fluorescence stability should be determined for each cell type and marker used, and the confounding effects of fixation on cell autofluorescence should be considered.     

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.     

Immunofluorescence analysis in flow cytometry: better selection of antibody-labeled cells after fluorescence overcompensation in the red channel

Selection of cells labeled with fluorescein isothiocyanate-conjugated (FITC) antibodies can be difficult if large autofluorescent cells are used and if the cells bind only a few molecules of antibody. We have developed a simple flow cytometric procedure that allows better selection of stained cells. When an argon ion laser emitting at 488 nm is used, the green fluorescence detected is the sum of cell autofluorescence and of the signal generated by the FITC antibody. Thus, when we subtract green signal from the red by fluorescence compensation, the signal of stained cells is on average reduced more than for the unstained counterpart. In this scenario, positive selection of cells with low red signal allows more efficient selection of stained cells. We tested the overcompensation procedure on mixtures of cells unstained and stained with a relevant FITC antibody. Cell mixtures were analyzed using normal vs increased levels of compensation in the red channel. Increased levels of compensation resulted in easier gating and higher recovery of stained cells. The efficiency of the overcompensation procedure was particularly high when using red filters with low cutoff (i.e., 560 or 570 nm), possibly because of the significant emission of fluorescein in the red channel, which caused separation between stained and unstained cells also in the red dimension. This method is useful for sorting cells expressing low levels of surface markers and facilitates selection of rare cells transfected with surface antigen genes. This technique is compatible with the use of propidium iodide for live/dead cell discrimination and with the subtraction of the cellular background of autofluorescence.     

Copper-mediated oxidative burst in Nicotiana tabacum L. cv. Bright Yellow 2 cell suspension cultures

In cell suspension cultures of Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) a rapid and concentration-dependent accumulation of H(2)O(2) is induced by excess concentrations of copper (up to 100 microM). This specific and early response towards copper stress was shown to be extracellular. Addition of 300 U of catalase per ml decreased the level of H(2)O(2). Superoxide dismutase (5 U/ml) induced an increase in H(2)O(2) production by 22.2%. This indicates that at least part of the H(2)O(2) is produced by dismutation of superoxide. Pretreatment of the cell cultures with the NAD(P)H oxidase inhibitors diphenylene iodonium (2 and 10 microM) and quinacrine (1 and 5 mM) prevented the generation of H(2)O(2) under copper stress for 90%. The influence of the pH on the H(2)O(2) production revealed the possible involvement of cell-wall-dependent peroxidases in the generation of reactive oxygen species after copper stress.     

Detection by flow cytometry of protoplast fusion and transient expression of transferred heterologous CD4 sequences in COS-7 cells

Transfer and expression of a plasmid containing the gene encoding the human T-cell antigen CD4 by protoplast fusion was measured by flow cytometry (FCM). Protoplasts were prelabeled with fluorescein isothiocyanate (FITC) and fused to COS-7 cells. Nonspecific protoplast adsorption to the plasma membrane was differentiated from successful protoplast fusion by the addition of an antibody directed against fluorescein to quench extracellular protoplast fluorescence. Transfection efficiencies were defined as both percent CD4 expressing cells and CD4 expression levels on a single cell basis in the transient immunofluorescence assay. Cell sorting studies indicated that intracellular protoplast-associated fluorescence immediately after fusion exhibited a good correlation with transient CD4 transfection efficiencies as measured by indirect immunofluorescence. Reconstruction experiments comparing CD4 transfer efficiencies of protoplast fusion and calcium phosphate transfection showed that fusion resulted in a higher percentage of CD4 expressing transfectants, while calcium phosphate transfection yielded higher CD4 expression levels on a single cell basis. Thus, FCM appears to be useful as a new tool for sensitive detection of transient expression of heterologous reporter genes in COS-7 cells.     

Rhodamine immunohistofluorescence applied to plant tissue.

Tissue slices from the roots and seeds of sanifoin (Onobrychis viciifolia, Scop.) exhibit bright autofluorescence when illuminated with blue (495 nm) light. This autofluorescence is indistinguishable from the fluorescence emission of fluorescein, the commonly used fluorochrome in immunohistochemical staining procedures. Rhodamine isothiocyanate, when coupled to immunoglobulin, and excited with green light at 546 nm, exhibits a reddish-orange fluorescence with an emission maximum at 590 nm. Plant tissue has little or no autofluorescence when illuminated at this wavelength and viewed with a 580 nm barrier filter. Therefore, use of rhodamine for immunohistochemical localization in plant tissue avoids interpretative complications due to inherent autofluorescence.     

The effect of tetrathiomolybdate on the metabolism of copper by hepatocytes and fibroblasts

Tetrathiomolybdate (TTM) has been examined for its effect on copper metabolism in mouse hepatocytes in primary culture and human fibroblasts. It decreased the amount of copper inside hepatocytes, decreased the rate of copper uptake by hepatocytes in a concentration dependent manner, and increased the copper efflux from the cells. TTM appeared to remove copper preferentially from the labile pool, but with a lower affinity than cage chelators. In fibroblasts, TTM only had a marginal effect on copper levels below a concentration of 100 microM and had no clear effect on the rate of copper uptake. TTM was not toxic to human fibroblasts, but in some preparations, a concentration of more than 50 microM was toxic to hepatocytes.     

Flow cytometry for the development of biotechnological processes with microalgae

The current interest in microalgae as a sustainable source of next generation biofuels and other valuable substances is driving exploration of their use as unique biotechnological production systems. To design and optimise appropriate production strategies, the behaviour of particular microalgal species should be well characterised under different culture conditions. Thus, flow cytometric (FCM) methods, which are already well established in environmental and toxicological studies of microalgae, are also useful for analysing the physiological state of microalgae, and have the potential to contribute to the rapid development of feasible bioprocesses. These methods are commonly based on the examination of intrinsic features of individual cells within a population (such as autofluorescence or size). Cells possessing the desired physiological or morphological features, which are detectable with or without fluorescent staining, are counted or isolated (sorted) using an FCM device. The options for implementation of FCM in the development of biotechnological processes detailed in this review are (i) analysing the chemical composition of biomass, (ii) monitoring cellular enzyme activity and cell viability, and (iii) sorting cells to isolate those overproducing the target compound or for the preparation of axenic cultures.     

Effect of copper sulfate on performance of a serum-free CHO cell culture process and the level of free thiol in the recombinant antibody expressed

A recombinant Chinese hamster ovary (CHO) cell line was used to express a humanized antibody. Product quality analysis of this humanized antibody showed the presence of free thiol, due to unpaired cysteine residues in the Fab region. Decreased potency of this thiol Fab made it critical to minimize the levels of free thiol. In an effort to do this, we evaluated the effect of copper sulfate addition to the cell culture production medium. As a component of the production medium, copper sulfate can act as an oxidizing agent, thereby facilitating disulfide bond formation. Four concentrations of copper sulfate were added at the beginning of 2-L benchtop production cultures of the recombinant CHO cell line: 0, 5, 50, and 100 microM. We found that these copper sulfate additions had no effect on cell growth or antibody production. However, a slight dose-dependent depression in culture viability was observed. Analysis of the purified antibody showed that either the 50 or 100 microM copper sulfate additions reduced the level of free thiol by more than 10-fold.     

Traces of copper ions deplete glutathione in human hepatoma cell cultures with low cysteine content

BACKGROUND: Cell death induced by intracellular glutathione depletion has been reported to be dependent on the presence of trace amounts of extracellular copper ions. Since little is known about the relationship between glutathione depletion and copper homeostasis, we have in the present study further investigated the role of low amounts of copper ions in glutathione depletion. METHODS: Glutathione turnover was investigated in HeLa and hepatoma cell cultures with normal and low cysteine content in the presence of copper ions (1 and 10micromol/L) and two other glutathione-stimulating agents (lipoic acid and mercury ions). RESULTS: Copper ions (10micromol/L) caused relatively small increases in total amount of glutathione (the sum of the intracellular and the extracellular amount of glutathione) in HeLa and hepatoma cell cultures with normal cysteine levels (420nmol/mL) compared to control cell cultures, whereas lipoic acid and mercury ions strongly increased total glutathione in both types of cell cultures. Lower amount of total glutathione was observed in cell cultures with a lower cysteine levels (84nmol/mL), which is similar to that in human plasma. A strongly decreased total amount of glutathione in the presence of copper ions was observed in hepatoma cell cultures with lower cysteine levels, whereas the other agents showed effects similar to those described for cell cultures with normal cysteine levels. CONCLUSION: Glutathione synthesis in hepatoma cell cultures is probably more sensitive to a low cysteine level than HeLa cell cultures, and the presence of copper ions further decreases the availability of cysteine probably by increasing the disulfide binding to cysteine residues in extracellular proteins, which causes a further decrease of total glutathione.     

Cellular responses to environmental contaminants in amoebic cells of the slime mould Dictyostelium discoideum

Amoebic Dictyostelium discoideum cells were employed in a bioassay to evaluate stress responses after exposures to the polyaromatic hydrocarbon benzo[a]pyrene (B[a]P) and two heavy metals (copper and mercury). Furthermore, we developed a recombinant cell line expressing a labile Green Fluorescent Protein (GFP) variant expressed under the control of an actin promoter to monitor stress-related protein degradation. Finally, cell viability was monitored to discriminate lethal exposure concentrations. The results demonstrated that exposure to sub-micromolar concentrations of mercury rendered significant changes in all studied physiological parameters, whereas B[a]P became toxic at low micromolar, and copper at high micromolar concentrations. Exposure to 0.5 microM mercury significantly reduced lysosomal membrane stability (LMS), endocytosis rate, GFP expression, and further resulted in the elevation of cytosolic free Ca(2+) ([Ca(2+)](i)). LMS in mercury-treated cells that had been pre-incubated with a specific Ca(2+)-dependent phospholipase A2 blocking agent was however not affected by the exposure, indicating that the toxic action of mercury is linked to the activation of phospholipase A2 via a Ca(2+)-signaling pathway. Exposure to 20 microM B[a]P significantly reduced LMS, endocytosis rate, and GFP expression, however without affecting [Ca(2+)](i), suggesting a calcium-independent route of toxicity for this compound. None of the physiological parameters were significantly affected by copper exposure at concentrations <400 microM, demonstrating a high resistance to this metal. Our results further showed that neither cell growth nor viability was affected by concentrations altering the studied physiological parameters. LMS, endocytosis rate, and [Ca(2+)](I), therefore, appear sensitive biomarkers of pollutant-related stress in amoebic cells.     

Long wavelength fluorophores and cell-by-cell correction for autofluorescence significantly improves the accuracy of flow cytometric energy transfer measurements on a dual-laser benchtop flow cytometer

BACKGROUND: Flow cytometric fluorescence resonance energy transfer (FCET) is an efficient method to map associations between biomolecules because of its high sensitivity to changes in molecular distances in the range of 1-10 nm. However, the requirement for a dual-laser instrument and the need for a relatively high signal-to-noise system (i.e., high expression level of the molecules) pose limitations to a wide application of the method. METHODS: Antibodies conjugated to cyanines 3 and 5 (Cy3 and Cy5) were used to label membrane proteins on the cell surface. FCET measurements were made on a widely used benchtop dual-laser flow cytometer, the FACSCalibur, by using cell-by-cell analysis of energy transfer efficiency.ResultsTo increase the accuracy of FCET measurements, we applied a long wavelength donor-acceptor pair, Cy3 and Cy5, which beneficially affected the signal-to-noise ratio in comparison with the classic pair of fluorescein and rhodamine. A new algorithm for cell-by-cell correction of autofluorescence further improved the sensitivity of the technique; cell subpopulations with only slightly different FCET efficiencies could be identified. The new FCET technique was tested on various direct and indirect immunofluorescent labeling strategies. The highest FCET values could be measured when applying direct labeling on both (donor and acceptor) sides. Upon increasing the complexity of the labeling scheme by introducing secondary antibodies, we detected a decrease in the energy transfer efficiency. CONCLUSIONS: We developed a new FCET protocol by applying long wavelength excitation and detection of fluorescence and by refining autofluorescence correction. The increased accuracy of the new method makes cells with low receptor expression amenable to FCET investigation, and the new approach can be implemented easily on a commercially available dual-laser flow cytometer, such as a FACSCalibur.     

Tyrosine phosphorylation following alterations in arteriolar intraluminal pressure and wall tension

Arterioles respond to increased transmural pressure with myogenic constriction. The present study investigated the role of tyrosine phosphorylation in myogenic activity. Cannulated segments of a rat cremaster arteriole were fixed under pressure, followed by incubation with fluorescein isothiocyanate (FITC)-conjugated anti-phosphotyrosine. Smooth muscle cell fluorescence intensity was measured with the use of confocal laser-scanning microscopy. Anti-phosphotyrosine fluorescence intensity in muscle cells of arterioles maintained at 100 mmHg was reduced by the tyrosine kinase inhibitor tyrphostin A47 (30 microM) and increased by the tyrosine phosphatase inhibitor pervanadate (100 microM). In time-course experiments, anti-phosphotyrosine fluorescence increased slowly (over 5 min) after an acute increase in intraluminal pressure, and was dissociated from myogenic contraction (within 1 min). In contrast, angiotensin II (0.1 microM) caused rapid constriction and increased tyrosine phosphorylation. Anti-phosphotyrosine fluorescence was also pressure dependent (10-100 mmHg). Abolition of myogenic activity, either through removal of extracellular Ca2+, or exposure to verapamil (5 microM) or forskolin (0.1 microM) caused a further increase in anti-phosphotyrosine fluorescence. We conclude that transmural pressure and/or wall tension in arterioles causes increased tyrosine phosphorylation; however, this is not involved in the acute phase of myogenic constriction but may be involved in later responses, such as sustained myogenic tone or mechanisms possibly related to growth.     

Quantitative evaluation of the phagocytic activity of endothelial cells in culture

The phagocytic activity of endothelial cells (EC) from human umbilical vein was analysed quantitatively in primary culture. EC were incubated with fluorescent carboxylated microspheres (FCM) and the intensity of fluorescence was measured on spectrofluorimeter. It was found that EC phagocyted actively only FCM of limited diameter (not more than 0.26 mu). The most intensive phagocytosis occurred in the first 60 min of the experiment, the following incubation with FCM did not influence significantly the number of phagocyted particles but increased nonspecific binding. High doses of FCM stimulated phagocytosis within EC. The phagocytic activity of EC depended on the growth stage: it was maximum in proliferated cells and sharply decreased in confluent cultures. This method may be useful for the comparison of phagocytic activity of different cell types, as well as for drug testing.     

Fluorescence correlation microscopy of cells in the presence of autofluorescence.

Fluorescence correlation microscopy (FCM), the combination of fluorescence correlation spectroscopy (FCS) and digital microscopy (Brock and Jovin, 1998. Cell. Mol. Biol. 44:847-856), has been implemented for measuring molecular diffusion and association in living cells with explicit consideration of autocorrelations arising from autofluorescence. Autofluorescence excited at 532 nm colocalizes with mitochondria, has flavin-like spectral characteristics, exhibits relaxation times characteristic for the diffusion of high-molecular-weight proteins, and depends on the incubation conditions of the cells. These time- and location-dependent properties preclude the assignment of universal background parameters. The lower limit for detection of microinjected dextran molecules labeled with the carboxymethylindocyanine dye Cy3 was a few thousand molecules per cell, and the diffusion constant of 1.7 x 10(-7) cm2/s agreed well with values measured with other methods. Based on the fluorescence signal per molecule (fpm) and the molecule number derived from autocorrelation analysis, a new method is devised to define intracellular association states. We conclude that FCM is a powerful, noninvasive method for probing molecular interactions in femtoliter volume elements within defined subcellular locations in living cells.     

Pharmacological levels of copper exert toxic effects in Caco-2 cells

Copper might be toxic to human intestinal cells because of its ability to catalyze the formation of free radicals. The aim of the present study was to quantify toxicological effects of increasing copper concentrations in preconfluent, colonic cancerous cells as well as in postconfluent, differentiating Caco-2 cells. Our results indicate that postconfluent cells might be more sensitive to copper toxicity. A significant rise of lactate dehydrogenase (LDH) release (150 μM or above) and decrease of cell proliferation (100 μM or above) with increasing copper levels was found, as compared to the control. To the contrary, preconfluent cells were not significantly affected by copper (LDH release) or, if so, only at a concentration of 250 μM (proliferation). Loss of viability and morphological changes, including loss of adherence and cell rounding, were visible after incubation with 250 μM copper in both groups. Superoxide dismutase (SOD) activities were not affected by copper. Glutathione peroxidase (GSH-Px) and catalase activities were higher in copper-treated cells, especially in the postconfluent ones (nevertheless, the results were not significant because of high standard deviations). In conclusion, we demonstrated that copper exerts intracellular, toxicological effects on both groups of Caco-2 cells, although the effects seem to be more evident in the postconfluent (enterocytelike) group. Risk assessment, especially for high concentrations, might be of special interest.