Journal of lmmunological Methods, 156 (1992) 39-45
39
© 1992 Elsevier Science Publishers B.V. All rights reserved 0022-1759/92/$05.00
JIM 06486
A microplate assay for the detection of oxidative products using 2',7'-dichlorofluorescin-diacetate * Alexander R. Rosenkranz, Sabine Schmaldienst, Karl M. Stuhlmeier, Wanjun Chen, Walter Knapp and G e r h a r d J. Zlabinger Institute of lmmunology, University of Vienna, A-1090 Vienna, Austria (Received 2 March 1992, revised received 7 April 1992, accepted 28 May 1992)
A fluorometric microplate assay was established for the detection of respiratory burst activity in phagocytic cells by assessing oxidation of 2',7'-dichlorofluorescin-diacetate (DCFH-DA). This method is based on flow cytometric studies by Bass et al. (J. Immunol. 130 (1983) p. 1910) describing intracellular detection of DCFH oxidation due to the presence of hydrogen peroxides. In the present study we have adapted the assay for use in microtiter plates to determine the amount of extracellular reactive oxidative products. DCFH-DA, granulocytes and stimuli (phorbol myristate acetate, n-formyl-methionyl-leucylphenylalanine, concanavalin A) were added to microtiter plates and after incubation at 37°C, the development of fluorescence intensity was read in a fluorescence concentration analyzer (FCA, Baxter). Calibration of fluorescence units recorded by the FCA was achieved by comparison with defined amounts of fluorescent DCF. The change in measured fluorescence was linear with cell density over the range of 2 x 10s-1 x 106 cells/well. Cumulative DCF generation in individual wells could be recorded non-destructively at frequent intervals for time course measurements. Results from FCA measurements correlated perfectly with the FACS analysis of the same samples (r = 0.99). In conclusion, this assay can be useful for screening monoclonal antibodies recognizing cell surface structures possibly involved in signal transduction as well as for testing phagocytes for their capacity to release reactive oxidative intermediates. Key words: 2',7'-Dichlorofluorescin-diacetate; Automated microplate assay; Respiratory burst; Phagocyte
Introduction
Release of toxic oxygen metabolites is regarded an essential component of phagocyte Correspondence to: G.J. Zlabinger, Institute of Immunology, Borschkegasse 8A, A-1090 Vienna, Austria. Tel.: 1431375/356; Fax: 1-4086670. * Supported by Medizinisch wissenschaftlicher Fonds des Biirgermeisters der Bundeshauptstadt Wien. Abbreviations: DCF, 2',7'-dichlorofluorescein; DCFH-DA, 2',7'-dichlorofluorescin-diacetate; FCA, fluorescence concentration analyzer; fMLP, n-formyl-methionyl-leucyl-phenylalanine; PMA, phorbol myristate acetate.
function in host defence against infections as well as in the pathogenesis of inflammatory processes. Since there is increasing evidence for the involvement of a variety of cell surface molecules on phagocytic cells in the induction of the respiratory burst (Crockett-Torabi and Fantone, 1990; Fischer et al., 1990; Macintyre et al., 1989; Nong et al., 1989; Ockenhouse et al., 1989; Stockinger et al., 1990; Trezzini et al., 1990), there is a need for an assay which can assess cell activation by measuring oxidative product formation in a large number of samples. Such an assay could be used for screening monoclonal antibodies recognizing
40 different cell surface structures on phagocytic cells, thus allowing additional information to be obtained about the mechanisms involved in the induction as well as in the effector phase of the respiratory burst. It has recently come to light that the capacity of phagocytes to secrete reactive oxygen intermediates is closely regulated by cytokines which either act as priming agents or can induce phagocyte activation by themselves (Steinbeck and Roth, 1989). Therefore, the role of cytokines could also be monitored by such a test system. Furthermore, measurement of the ability to produce reactive oxygen intermediates is of great importance for the diagnostic evaluation of acquired as well as inherited phagocyte dysfunction and would facilitate testing of substances with pro- or anti-inflammatory effects. In addition to several test systems using different indicators for the detection of respiratory burst products in phagocytic cells (Babior et al., 1973; De la Harpe and Nathan, 1985; Emmend6rffer et al., 1990; Fattorossi et al., 1990; Nowak, 1990; Pick, 1986; Root et al., 1975; Van Kessel et al., 1989), a method to measure intracellular H2Ozproduction has already proved useful in evaluating oxidative product formation in response to several stimuli at the single cell level (Bass et al., 1983). The authors utilized 2',7'-dichlorofluorescin-diacetate (DCFH-DA), a non polar compound that readily diffuses into cells. It is hydrolyzed by intracellular esterases to the nonfluorescent derivative 2',7'-dichlorofluorescin, which is polar and trapped within the cells. In the presence of intracellular hydrogen peroxide, this compound is oxidized to the highly fluorescent co mp o u nd 2',7'-dichlorofluorescein (DCF). Cellular fluorescence intensity was shown by Bass and co-workers to be directly proportional to the level of DCF formed intracellularly and monitored by flow cytometry. This method is based on a fluorimetric assay previously described by Keston and Brandt (1965), who employed DCFH-DA to measure hydrogen peroxide in aqueous solution. In the presence of peroxidase, oxidation of the nonfluorescent reagent could be attained by picomoles of hydrogen peroxide with a linear relationship between peroxide concentration and detected fluorescence. Assessment of oxidative product formation
within cells is based on the assumption that, due to its polarity, fluorescent DCF remains intracellular (Bass et al., 1983; Hirabayashi et al., 1985). This, however, does not exclude the formation of extracellular reactive oxidative products which might cause the conversion of DCFH-DA to its fluorescent counterpart in supernatants. Here, we present a modification of the method described by Bass et al. (1983) to measure respiratory burst activity in an automatic fluorometric microplate reader determining the amount of DCF formed after stimulation of phagocytic cells. Using this assay, cumulative oxidant formation may be recorded non-destructively in a great number of samples at frequent time intervals for time course measurements.
Materials and methods
Reagents Phorbol myristate acetate (PMA), n-formylmethionyl-leucyl-phenylalanine (fMLP), concanavalin A (ConA) and 2',7'-dichlorofluorescein (DCF) were obtained from Sigma Chemicals (St. Louis, MO, USA). 2',7'-dichlorofluorescin-diacetate was purchased from Molecular Probes (Junction City, OR, USA). DCFH-DA was dissolved in ethanol at a concentration of 2.5 mg/ml, whereas fMLP and PMA were dissolved in DMSO at a concentration of 10-2 M, and stored in the dark at -20°C. Stock solutions of DCF were prepared in ethanol (25 mM).
Preparation of granulocytes Heparinized whole blood from healthy volunteers was centrifuged on a Ficoll-Paque density gradient (Pharmacia, Uppsala, Sweden). After separation, granulocytes were isolated from the pellet by lysis of red blood cells using an ammonium chloride buffer. Cells were washed twice in saline, resuspended in phosphate-buffered saline (PBS) containing calcium (0.9 mM) and magnesium (0.4 mM) to give a concentration of 2 x 107/ml and kept on ice until use.
Assay of oxidative product formation Respiratory burst was assessed by measuring oxidation of DCFH-DA with a spectrofluorome-
41
ter (wavelength 485/535 nm) capable of reading microtiter plates (fluorescence concentration analyzer, FCA, Baxter, Mundelein, IL, USA). The instrument was calibrated using a multi-wavelength reference assay plate with fluorescent particles incorporated into the membrane on the bottom of each well. Well-to-well precision of the fluorometer was determined and variation was less than 8% CV for the respective wavelength. For comparison, intracellular oxidative product formation was determined on a flow cytometer (FACScan, Sunnyvale, CA, USA) as described previously (Bass et al., 1983). Equal volumes (50/xl) of granulocytes, DCFHDA and known inducers of oxidative product formation were added to flat bottom microtiter plates (Costar). Unless otherwise indicated DCFH-DA was used at a final concentration of 10 /zg/ml or 1 /xg/ml. PMA (10-6-10 -1° M), fMLP (10-6-10 -1° M) and ConA ( 5 - 2 0 / z g / m l ) served as stimuli. During preparation of the assay all reagents were kept at 4°C. Then the microtiter plates were transferred to a water bath and incubated at 37°C for the indicated time periods. Measurement of developing fluorescence intensity was performed every 15 min on the FCA. In experiments comparing FCA and FACS analysis, plates were read after 15 and 30 min on the
spectrofluorometer and then put on ice. Samples were transferred to polystyrene tubes (Falcon 2052, Lincoln Park, N J, USA) and immediately analyzed on a FACScan. Granulocytes were gated according to forward and side scatter, and fluorescence intensity was recorded on a logarithmic scale.
Results
Kinetic studies of DCF generation As an expression of oxidative product formation, the conversion of nonfluorescent DCFH-DA to its fluorescent counterpart was assessed in the culture supernatants of stimulated granulocytes. After incubation of granulocytes with PMA (10 -6 M), fMLP ( 1 0 - 6 M), ConA (20 /zg/ml) or with PBS, fluorescence intensity (FI) of the samples was determined every 5 min. We found a continuous increment in stimulated cultures whereas only a small increase of fluorescence was observable in control cultures. Using various concentrations of DCFH-DA (0.3-10 /zg/ml), differences in FI between cultures incubated with weaker stimuli such as fMLP (10 -6 M) or ConA and control cultures were more pronounced when DCFH-DA had been added at a concentration of 1 / z g / m l or 2000
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Fig. 1. Relationship between cell count and the formation of oxidative burst products. After incubation for 30 min at 37°C fluorescence intensity ( A ) or attomoles D C F / c e l l (B) obtained from stimulated and unstimulated cells were assessed at increasing concentrations ( 2 - 1 0 x l 0 5 / w e l l ) . A linear relationship between cell count and generation of fluorescent D C F was observed. D C F H - D A concentrations of 10 and 1 p , g / m l were used.
42 even less. In contrast, stimulation with P M A required higher concentrations of D C F H - D A (10 /zg/ml) since otherwise the amount of the indicator appeared to become limiting for the detection of oxidative product formation (data not shown). A linear relationship between cell Count and the amount of oxidative burst product formation was established. Increasing numbers of granulocytes (2 × 106-2 × 107 cells/ml) were stimulated with P M A (10 -6 M) and incubated for 30 min at 37°C (Fig. 1). The change in fluorescence intensity was linear over the cell concentrations tested if D C F H - D A was used at a concentration of 10 /xg/ml. At 1 /xg/ml D C F H - D A fluorescence intensity leveled off at a higher cell density apparently because of the relative lack of nonfluorescent dye.
(Bass et al., 1983), we calibrated our assay by measuring the fluorescence intensity of definite amounts of DCF. Fluorescence of D C F was shown to be a constant and reproducible standard by measuring serial dilutions of 2',7'-dichlorofluorescein at different times (Fig. 2). 1 day after preparing the dilutions a slight but insignificant decrease in fluorescence intensity was detected. When dilutions of D C F were prepared on different days only minor differences in fluorescence intensity were found. Using this standard curve of D C F we were able to transform fluorescence intensity measured in stimulated or unstimulated cultures into attomoles (amol) DCF/ceI1.
Characterization of the cellular compartment responsible for DCFH-DA oxidation In order to characterize the cellular compartment responsible for D C F generation as measured by the spectrofluorometer, the detection of cell-associated and extracellular fluorescent dye
Calibration of the DCFH-DA assay Since it has been established that the fluorescent oxidation product of D C F H - D A was D C F 100000 H
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Fig. 2. Calibration of fluorescence concentrations measured on the FCA. Fluorescence intensity of serial dilutions of fluorescent DCF was assessed at different times: (1) immediately after preparation, (2) 30 min and (3) 60 min after preparation, (4) on day 2. On day 2 also dilutions from the stock solution of day 1 were prepared and measured directly after preparation (5) as well as 30 min later (6). Further, on day 2 a new stock solution and serial dilutions thereof were prepared and measured immediately after preparation (7) and after 30 min (8).
43 was evaluated on the FCA. After stimulation with P M A for 30 min the reaction mixture as well as the supernatant thereof and the granulocytes which had been washed three times were analyzed separately. The fluorescence intensity of the supernatant was as high as that of the unseparated sample and only a low cell-associated signal was detected by the FCA. However, we clearly detected oxidation of D C F H - D A within those granulocytes by FACS analysis (data not shown). Thus, the FCA appears to assess extracellular reactive oxidative products whereas the FACS measures intracellular burst formation.
~
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Comparison between FACS and FCA Since DCF generation is assessed in different cellular compartments by FCA and FACS, we also examined oxidative product formation in the same sample by the microplate assay and the flow cytometric method in order to investigate the comparability of the test systems. After stimula-
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day of investigation Fig. 4. Inter- and intraindividual variations in the formation of oxidative products. On four successive days (1-4) stimulation of granulocytes with P M A 10 -6 M (closed symbols) and C o n A 20 p . g / m l (open symbols) was performed in three
/
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tion with different inducers of the respiratory burst (PMA 10-6-10 -8 M; fMLP 10-6-10 -8 M; ConA 5 - 2 0 # g / m l ) , evaluation b y both assays showed an excellent correlation (r = 0.99). In addition, we also demonstrated that FACS and FCA were equally sensitive in the detection of oxidative products as reflected by the slope (k = 0.98) of the regression curve (Fig. 3).
r o 0,99 k = 0.98 d = 0.28
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different individuals (AR, GZ, GB). Release of oxidative burst products is expressed as a stimulation index. Different symbols refer to the individuals tested.
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Inter- and intraindividual variations in the forma.tion of oxidative products
0 0
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SI/FACS
Fig. 3. Correlation of D C F m e a s u r e m e n t by flow cytometry and spectrofluorometry. Stimulated ( P M A . 1 0 6 to 10 - 8 M, fMLP 10 - 6 to 10 - 8 M, C o n A 5 - 2 0 / x g / m l ) and unstimulated samples were investigated after 30 min incubation at 37°C both on F C A and FACS. A correlation between these two evaluation systems was established by comparison of stimulation indices (SI) obtained on FACS and FCA. SI were calculated according to the following formula: SI = fluorescence intensity of stimulated c u l t u r e s / f l u o r e s c e n c e intensity of controls. The small insert in the left upper corner shows the correlation for the weak stimuli (fMLP, ConA).
Using granulocytes from three healthy individuals, extracellular DCF concentrations were measured on the FCA on four different days in order to determine inter- an~l intraindividual variations in the formation of oxidative products. Both spontaneous and stimulated release of reactive oxygen intermediates differed considerably between individuals. Variations in oxidative product formation within one individual as well as the relationship between individuals when tested on different days appeared to be relatively constant (Fig. 4).
44
Discussion Here, we describe a rapid and simple fluorometer-based microplate assay which permits the evaluation of oxidative product formation in a great number of samples within a reasonable time and with an appropriate expenditure of effort. Using this method, extracellular reactive oxygen intermediates can be assessed as demonstrated by the presence of fluorescent dye in culture supernatants. It has been stated previously (Bass et al., 1983) that the deesterified indicator reagent is trapped within cells and, therefore, in our system, oxidation of the nonfluorescent dye must have occurred extracellularly. We also found that after loading of PMN with DCFH-DA and subsequent washing, the fluorescent dye generated after stimulation with PMA, could be detected by FACS but not by the FCA (data not shown). Comparing the results from our test system with those obtained from the previously established flow cytometric assay (Bass et al., 1983), which used the same indicator reagent, demonstrates that both methods are equally capable of assessing the ability of phagocytes to mount a respiratory burst response. It is not possible to dissect the production of reactive oxygen intermediates between subpopulations in our microplate assay as was the case with the fluorometer. However, the assay has the great advantage that samples can be evaluated simultaneously and do not have to be processed separately. The present test system represents a useful tool for screening samples for oxidative product formation. If it is necessary to evaluate particular probes in more detail, the use of the same indicator reagent would permit the measurement of selected samples by flow cytometry also. Samples can be measured in a very short time and therefore, it is possible to investigate dose response relationships as well as perform strictly timed inhibition tests. Moreover, fluorescence measurements in microplates are non destructive and multiple readings can be undertaken on single cultures. Therefore it is also possible to make kinetic studies after stimulation with a potential agonist. Microplate assays have been described using ferricytochrome C, phenol red (Pick and Mizel,
1981), nitroblue tetrazolium (Pick, 1986) and scopoletin (De la Harpe and Nathan, 1985). The DCFH-DA based microassay offers the advantage over these test systems that fluorescence concentration increases with ongoing respiratory burst activity. However, the formation of oxidative products in those assays, is indicated by a decrease in a particular signal and is consequently associated with lower sensitivity. This applies both for scopoletin which after oxidation becomes nonfluorescent and indirectly also for the photometric measurement of ferricytochrome C or nitroblue tetrazolium since determination of absorbances insures measuring the diminution of light induced by a particular dye. Moreover, the differences in absorbance values in microplate assays using ferricytochrome C are often small sometimes making it difficult to clearly differentiate between stimulated cultures and controls (Pick and Mizel, 1981). Furthermore, unlike the scopoletin assay which requires added peroxidase, possible interference from reducing co-substrates is minimized in the DCFH-DA test. In conclusion, we have adapted a fluorometric burst assay for use in microtiter plates which permits reproducible detection of extracellular reactive oxygen intermediates. This technique should help to further elucidate the mechanisms of the respiratory burst response providing the facility for large scale evaluation of the role of surface receptors on phagocytes as well as for testing the effect of pro- and antiinflammatory substances. Since strong evidence exists for the extracellular release of superoxide anions and its dismutation product hydrogen peroxide in inflammatory processes such as the adult respiratory distress syndrome (Malech and Gallin, 1987), this assay, determining extracellular reactive oxygen intermediates, may provide a valuable addition to existing methods which assess intracellular burst formation.
References Babior, B.M., Kipnes, R.S. and Curnutte, J.T. (1973) Biological defense mechanisms. The production by leucocytes of superoxide, a potential bactericidal agent. J. Clin. Invest. 52, 741.
45 Bass, D.A., Parce, J.W., Dechatelet, L.R., Szejda, P., Seeds, M.C. and Thomas, M. (1983) Flow cytometric studies of oxidative product formation by neutrophils: a graded response to membrane stimulation. J. Immunol. 130, 1910. Crockett-Torabi, E. and Fantone, J.C. (1990) Soluble and insoluble immune complexes activate human neutrophil NADPH oxidase by distinct Fc3, receptor-specific mechanisms. J. Immunol. 145, 3026. De la Harpe, J. and Nathan, C.F. (1985) A semi-automated micro-assay for H 2 0 2 release by human blood monocytes and mouse peritoneal macrophages. J. Immunol. Methods 78, 323. Emmend6rffer, A., Hecht, M., Lohmann-Matthes, M.-L. and Roesler, J. (1990) A fast and easy method to determine the production of reactive oxygen intermediates by human and murine phagocytes using dihydrorhodamine 123. J. Immunol. Methods 131, 269. Fattorossi, A., Nisini, R., Le Moli, S., De Petrillo, G. and D'Amelio, R. (1990) Flow cytometric evaluation of nitro blue tetrazolium (NBT) reduction in human polymorphnuclear leukocytes. Cytometry 11, 907. Fischer, G.F., Majdic, O., Gadd, S. and Knapp, W. (1990) Signal transduction in lymphocytic and myeloid cells via CD24, a new member of phosphoinositol-anchored membrane molecules. J. Immunol. 144, 638. Hirabayashi, Y., Taniuchi, S. and Kobayashi, Y. (1985) A quantitative assay of oxidative metabolism by neutrophils in whole blood using flow cytometry. J. Immunol. Methods 82, 253. Keston, A.S. and Brandt, R. (1965) The fluorometric analysis of ultramicro quantities of hydrogen peroxide. Anal. Biochem. 11, 1. Macintyre, E.A., Roberts, P.J., Jones, M., Van der Schoot, C.E., Favalaro, E.J., Tidman, N. and Linch, D.C. (1989) Activation of human monocytes occurs on cross-linking monocytic antigens to an Fc receptor. J. Immunol. 142, 2377. Malech, H.L. and Gallin, J.I. (1987) Neutrophils in human diseases. New Engl. J. Med. 317, 687. Nong, Y.-H., Remold-O'Donnell, E., Lebien, T.W. and Remold, H.G. (1989) A monoclonal antibody to sialophorin
(CD43) induces homotypic adhesion and activation of human monocytes. J. Exp. Med. 170, 259. Nowak, D. (1990) Hydrogen peroxide release from human polymorphnuclear leukocytes measured with horseradish peroxidase and o-dianisidine. Effect of various stimulators and cytochalasin B. Biomed. Biochim. Acta 49, 353. Ockenhouse, C.F., Magowan, C. and Chulay, J.D. (1989) Activation of monocytes and platelets by monoclonal antibodies or malaria-infected erythrocytes binding to the CD36 surface receptor in vitro. J. Clin. Invest. 84, 468. Pick, E. (1986) Microassays for superoxide and hydrogen peroxide production and nitroblue tetrazolium reduction using an enzyme immunoassay microplate reader. Methods Enzymol. 132, 407. Pick, E. and Mizel, D. (1981) Rapid microassays for the measurement of superoxide and hydrogen peroxide production by macrophages in culture using an automatic enzyme immunoassay reader. J. Immunol. Methods 46, 211. Root, R.K., Metcalf, J., Oshino, N. and Chance, B. (1975) H 2 0 2 release from human granulocytes during phagocytosis. I. Documentation, quantitation, and some regulating factors. J. Clin. Invest. 55, 945. Steinbeck, A.M. and Roth, J.A. (1989) Neutrophil activation by recombinant cytokines. Rev. Infect. Dis. 11,549. Stockinger, H., Gadd, S.J., Eher, R., Majdic, O., Schreiber, W., Kasinrerk, W., Strass, B., Schnabl, E. and Knapp, W. (1990) Molecular characterization and functional analysis of the leukocyte surface protein CD31. J. Immunol. 145, 3889. Trezzini, C., Jungi, T.W., Spycher, M.O., Maly, F.E. and Rap, P. (1990) Human monocytes CD36 and CD16 are signalling molecules. Evidence from studies using antibodyinduced chemiluminescence as a toll to probe signal transduction. Immunology 71, 29. Van Kessel, K.P.M., Van Strijp, J.A.G., Miltenburg, L.M., Van Kats-Renaud, H.J., Fluit, A.C. and Verhoef, J. (1989) Antibody-coated target cell membrane-induced chemiluminescene by human polymorphnuclear leukocytes. J. Immunol. Methods 118, 279.
39
© 1992 Elsevier Science Publishers B.V. All rights reserved 0022-1759/92/$05.00
JIM 06486
A microplate assay for the detection of oxidative products using 2',7'-dichlorofluorescin-diacetate * Alexander R. Rosenkranz, Sabine Schmaldienst, Karl M. Stuhlmeier, Wanjun Chen, Walter Knapp and G e r h a r d J. Zlabinger Institute of lmmunology, University of Vienna, A-1090 Vienna, Austria (Received 2 March 1992, revised received 7 April 1992, accepted 28 May 1992)
A fluorometric microplate assay was established for the detection of respiratory burst activity in phagocytic cells by assessing oxidation of 2',7'-dichlorofluorescin-diacetate (DCFH-DA). This method is based on flow cytometric studies by Bass et al. (J. Immunol. 130 (1983) p. 1910) describing intracellular detection of DCFH oxidation due to the presence of hydrogen peroxides. In the present study we have adapted the assay for use in microtiter plates to determine the amount of extracellular reactive oxidative products. DCFH-DA, granulocytes and stimuli (phorbol myristate acetate, n-formyl-methionyl-leucylphenylalanine, concanavalin A) were added to microtiter plates and after incubation at 37°C, the development of fluorescence intensity was read in a fluorescence concentration analyzer (FCA, Baxter). Calibration of fluorescence units recorded by the FCA was achieved by comparison with defined amounts of fluorescent DCF. The change in measured fluorescence was linear with cell density over the range of 2 x 10s-1 x 106 cells/well. Cumulative DCF generation in individual wells could be recorded non-destructively at frequent intervals for time course measurements. Results from FCA measurements correlated perfectly with the FACS analysis of the same samples (r = 0.99). In conclusion, this assay can be useful for screening monoclonal antibodies recognizing cell surface structures possibly involved in signal transduction as well as for testing phagocytes for their capacity to release reactive oxidative intermediates. Key words: 2',7'-Dichlorofluorescin-diacetate; Automated microplate assay; Respiratory burst; Phagocyte
Introduction
Release of toxic oxygen metabolites is regarded an essential component of phagocyte Correspondence to: G.J. Zlabinger, Institute of Immunology, Borschkegasse 8A, A-1090 Vienna, Austria. Tel.: 1431375/356; Fax: 1-4086670. * Supported by Medizinisch wissenschaftlicher Fonds des Biirgermeisters der Bundeshauptstadt Wien. Abbreviations: DCF, 2',7'-dichlorofluorescein; DCFH-DA, 2',7'-dichlorofluorescin-diacetate; FCA, fluorescence concentration analyzer; fMLP, n-formyl-methionyl-leucyl-phenylalanine; PMA, phorbol myristate acetate.
function in host defence against infections as well as in the pathogenesis of inflammatory processes. Since there is increasing evidence for the involvement of a variety of cell surface molecules on phagocytic cells in the induction of the respiratory burst (Crockett-Torabi and Fantone, 1990; Fischer et al., 1990; Macintyre et al., 1989; Nong et al., 1989; Ockenhouse et al., 1989; Stockinger et al., 1990; Trezzini et al., 1990), there is a need for an assay which can assess cell activation by measuring oxidative product formation in a large number of samples. Such an assay could be used for screening monoclonal antibodies recognizing
40 different cell surface structures on phagocytic cells, thus allowing additional information to be obtained about the mechanisms involved in the induction as well as in the effector phase of the respiratory burst. It has recently come to light that the capacity of phagocytes to secrete reactive oxygen intermediates is closely regulated by cytokines which either act as priming agents or can induce phagocyte activation by themselves (Steinbeck and Roth, 1989). Therefore, the role of cytokines could also be monitored by such a test system. Furthermore, measurement of the ability to produce reactive oxygen intermediates is of great importance for the diagnostic evaluation of acquired as well as inherited phagocyte dysfunction and would facilitate testing of substances with pro- or anti-inflammatory effects. In addition to several test systems using different indicators for the detection of respiratory burst products in phagocytic cells (Babior et al., 1973; De la Harpe and Nathan, 1985; Emmend6rffer et al., 1990; Fattorossi et al., 1990; Nowak, 1990; Pick, 1986; Root et al., 1975; Van Kessel et al., 1989), a method to measure intracellular H2Ozproduction has already proved useful in evaluating oxidative product formation in response to several stimuli at the single cell level (Bass et al., 1983). The authors utilized 2',7'-dichlorofluorescin-diacetate (DCFH-DA), a non polar compound that readily diffuses into cells. It is hydrolyzed by intracellular esterases to the nonfluorescent derivative 2',7'-dichlorofluorescin, which is polar and trapped within the cells. In the presence of intracellular hydrogen peroxide, this compound is oxidized to the highly fluorescent co mp o u nd 2',7'-dichlorofluorescein (DCF). Cellular fluorescence intensity was shown by Bass and co-workers to be directly proportional to the level of DCF formed intracellularly and monitored by flow cytometry. This method is based on a fluorimetric assay previously described by Keston and Brandt (1965), who employed DCFH-DA to measure hydrogen peroxide in aqueous solution. In the presence of peroxidase, oxidation of the nonfluorescent reagent could be attained by picomoles of hydrogen peroxide with a linear relationship between peroxide concentration and detected fluorescence. Assessment of oxidative product formation
within cells is based on the assumption that, due to its polarity, fluorescent DCF remains intracellular (Bass et al., 1983; Hirabayashi et al., 1985). This, however, does not exclude the formation of extracellular reactive oxidative products which might cause the conversion of DCFH-DA to its fluorescent counterpart in supernatants. Here, we present a modification of the method described by Bass et al. (1983) to measure respiratory burst activity in an automatic fluorometric microplate reader determining the amount of DCF formed after stimulation of phagocytic cells. Using this assay, cumulative oxidant formation may be recorded non-destructively in a great number of samples at frequent time intervals for time course measurements.
Materials and methods
Reagents Phorbol myristate acetate (PMA), n-formylmethionyl-leucyl-phenylalanine (fMLP), concanavalin A (ConA) and 2',7'-dichlorofluorescein (DCF) were obtained from Sigma Chemicals (St. Louis, MO, USA). 2',7'-dichlorofluorescin-diacetate was purchased from Molecular Probes (Junction City, OR, USA). DCFH-DA was dissolved in ethanol at a concentration of 2.5 mg/ml, whereas fMLP and PMA were dissolved in DMSO at a concentration of 10-2 M, and stored in the dark at -20°C. Stock solutions of DCF were prepared in ethanol (25 mM).
Preparation of granulocytes Heparinized whole blood from healthy volunteers was centrifuged on a Ficoll-Paque density gradient (Pharmacia, Uppsala, Sweden). After separation, granulocytes were isolated from the pellet by lysis of red blood cells using an ammonium chloride buffer. Cells were washed twice in saline, resuspended in phosphate-buffered saline (PBS) containing calcium (0.9 mM) and magnesium (0.4 mM) to give a concentration of 2 x 107/ml and kept on ice until use.
Assay of oxidative product formation Respiratory burst was assessed by measuring oxidation of DCFH-DA with a spectrofluorome-
41
ter (wavelength 485/535 nm) capable of reading microtiter plates (fluorescence concentration analyzer, FCA, Baxter, Mundelein, IL, USA). The instrument was calibrated using a multi-wavelength reference assay plate with fluorescent particles incorporated into the membrane on the bottom of each well. Well-to-well precision of the fluorometer was determined and variation was less than 8% CV for the respective wavelength. For comparison, intracellular oxidative product formation was determined on a flow cytometer (FACScan, Sunnyvale, CA, USA) as described previously (Bass et al., 1983). Equal volumes (50/xl) of granulocytes, DCFHDA and known inducers of oxidative product formation were added to flat bottom microtiter plates (Costar). Unless otherwise indicated DCFH-DA was used at a final concentration of 10 /zg/ml or 1 /xg/ml. PMA (10-6-10 -1° M), fMLP (10-6-10 -1° M) and ConA ( 5 - 2 0 / z g / m l ) served as stimuli. During preparation of the assay all reagents were kept at 4°C. Then the microtiter plates were transferred to a water bath and incubated at 37°C for the indicated time periods. Measurement of developing fluorescence intensity was performed every 15 min on the FCA. In experiments comparing FCA and FACS analysis, plates were read after 15 and 30 min on the
spectrofluorometer and then put on ice. Samples were transferred to polystyrene tubes (Falcon 2052, Lincoln Park, N J, USA) and immediately analyzed on a FACScan. Granulocytes were gated according to forward and side scatter, and fluorescence intensity was recorded on a logarithmic scale.
Results
Kinetic studies of DCF generation As an expression of oxidative product formation, the conversion of nonfluorescent DCFH-DA to its fluorescent counterpart was assessed in the culture supernatants of stimulated granulocytes. After incubation of granulocytes with PMA (10 -6 M), fMLP ( 1 0 - 6 M), ConA (20 /zg/ml) or with PBS, fluorescence intensity (FI) of the samples was determined every 5 min. We found a continuous increment in stimulated cultures whereas only a small increase of fluorescence was observable in control cultures. Using various concentrations of DCFH-DA (0.3-10 /zg/ml), differences in FI between cultures incubated with weaker stimuli such as fMLP (10 -6 M) or ConA and control cultures were more pronounced when DCFH-DA had been added at a concentration of 1 / z g / m l or 2000
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Fig. 1. Relationship between cell count and the formation of oxidative burst products. After incubation for 30 min at 37°C fluorescence intensity ( A ) or attomoles D C F / c e l l (B) obtained from stimulated and unstimulated cells were assessed at increasing concentrations ( 2 - 1 0 x l 0 5 / w e l l ) . A linear relationship between cell count and generation of fluorescent D C F was observed. D C F H - D A concentrations of 10 and 1 p , g / m l were used.
42 even less. In contrast, stimulation with P M A required higher concentrations of D C F H - D A (10 /zg/ml) since otherwise the amount of the indicator appeared to become limiting for the detection of oxidative product formation (data not shown). A linear relationship between cell Count and the amount of oxidative burst product formation was established. Increasing numbers of granulocytes (2 × 106-2 × 107 cells/ml) were stimulated with P M A (10 -6 M) and incubated for 30 min at 37°C (Fig. 1). The change in fluorescence intensity was linear over the cell concentrations tested if D C F H - D A was used at a concentration of 10 /xg/ml. At 1 /xg/ml D C F H - D A fluorescence intensity leveled off at a higher cell density apparently because of the relative lack of nonfluorescent dye.
(Bass et al., 1983), we calibrated our assay by measuring the fluorescence intensity of definite amounts of DCF. Fluorescence of D C F was shown to be a constant and reproducible standard by measuring serial dilutions of 2',7'-dichlorofluorescein at different times (Fig. 2). 1 day after preparing the dilutions a slight but insignificant decrease in fluorescence intensity was detected. When dilutions of D C F were prepared on different days only minor differences in fluorescence intensity were found. Using this standard curve of D C F we were able to transform fluorescence intensity measured in stimulated or unstimulated cultures into attomoles (amol) DCF/ceI1.
Characterization of the cellular compartment responsible for DCFH-DA oxidation In order to characterize the cellular compartment responsible for D C F generation as measured by the spectrofluorometer, the detection of cell-associated and extracellular fluorescent dye
Calibration of the DCFH-DA assay Since it has been established that the fluorescent oxidation product of D C F H - D A was D C F 100000 H
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Fig. 2. Calibration of fluorescence concentrations measured on the FCA. Fluorescence intensity of serial dilutions of fluorescent DCF was assessed at different times: (1) immediately after preparation, (2) 30 min and (3) 60 min after preparation, (4) on day 2. On day 2 also dilutions from the stock solution of day 1 were prepared and measured directly after preparation (5) as well as 30 min later (6). Further, on day 2 a new stock solution and serial dilutions thereof were prepared and measured immediately after preparation (7) and after 30 min (8).
43 was evaluated on the FCA. After stimulation with P M A for 30 min the reaction mixture as well as the supernatant thereof and the granulocytes which had been washed three times were analyzed separately. The fluorescence intensity of the supernatant was as high as that of the unseparated sample and only a low cell-associated signal was detected by the FCA. However, we clearly detected oxidation of D C F H - D A within those granulocytes by FACS analysis (data not shown). Thus, the FCA appears to assess extracellular reactive oxidative products whereas the FACS measures intracellular burst formation.
~
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Comparison between FACS and FCA Since DCF generation is assessed in different cellular compartments by FCA and FACS, we also examined oxidative product formation in the same sample by the microplate assay and the flow cytometric method in order to investigate the comparability of the test systems. After stimula-
35"
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day of investigation Fig. 4. Inter- and intraindividual variations in the formation of oxidative products. On four successive days (1-4) stimulation of granulocytes with P M A 10 -6 M (closed symbols) and C o n A 20 p . g / m l (open symbols) was performed in three
/
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tion with different inducers of the respiratory burst (PMA 10-6-10 -8 M; fMLP 10-6-10 -8 M; ConA 5 - 2 0 # g / m l ) , evaluation b y both assays showed an excellent correlation (r = 0.99). In addition, we also demonstrated that FACS and FCA were equally sensitive in the detection of oxidative products as reflected by the slope (k = 0.98) of the regression curve (Fig. 3).
r o 0,99 k = 0.98 d = 0.28
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different individuals (AR, GZ, GB). Release of oxidative burst products is expressed as a stimulation index. Different symbols refer to the individuals tested.
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Inter- and intraindividual variations in the forma.tion of oxidative products
0 0
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Fig. 3. Correlation of D C F m e a s u r e m e n t by flow cytometry and spectrofluorometry. Stimulated ( P M A . 1 0 6 to 10 - 8 M, fMLP 10 - 6 to 10 - 8 M, C o n A 5 - 2 0 / x g / m l ) and unstimulated samples were investigated after 30 min incubation at 37°C both on F C A and FACS. A correlation between these two evaluation systems was established by comparison of stimulation indices (SI) obtained on FACS and FCA. SI were calculated according to the following formula: SI = fluorescence intensity of stimulated c u l t u r e s / f l u o r e s c e n c e intensity of controls. The small insert in the left upper corner shows the correlation for the weak stimuli (fMLP, ConA).
Using granulocytes from three healthy individuals, extracellular DCF concentrations were measured on the FCA on four different days in order to determine inter- an~l intraindividual variations in the formation of oxidative products. Both spontaneous and stimulated release of reactive oxygen intermediates differed considerably between individuals. Variations in oxidative product formation within one individual as well as the relationship between individuals when tested on different days appeared to be relatively constant (Fig. 4).
44
Discussion Here, we describe a rapid and simple fluorometer-based microplate assay which permits the evaluation of oxidative product formation in a great number of samples within a reasonable time and with an appropriate expenditure of effort. Using this method, extracellular reactive oxygen intermediates can be assessed as demonstrated by the presence of fluorescent dye in culture supernatants. It has been stated previously (Bass et al., 1983) that the deesterified indicator reagent is trapped within cells and, therefore, in our system, oxidation of the nonfluorescent dye must have occurred extracellularly. We also found that after loading of PMN with DCFH-DA and subsequent washing, the fluorescent dye generated after stimulation with PMA, could be detected by FACS but not by the FCA (data not shown). Comparing the results from our test system with those obtained from the previously established flow cytometric assay (Bass et al., 1983), which used the same indicator reagent, demonstrates that both methods are equally capable of assessing the ability of phagocytes to mount a respiratory burst response. It is not possible to dissect the production of reactive oxygen intermediates between subpopulations in our microplate assay as was the case with the fluorometer. However, the assay has the great advantage that samples can be evaluated simultaneously and do not have to be processed separately. The present test system represents a useful tool for screening samples for oxidative product formation. If it is necessary to evaluate particular probes in more detail, the use of the same indicator reagent would permit the measurement of selected samples by flow cytometry also. Samples can be measured in a very short time and therefore, it is possible to investigate dose response relationships as well as perform strictly timed inhibition tests. Moreover, fluorescence measurements in microplates are non destructive and multiple readings can be undertaken on single cultures. Therefore it is also possible to make kinetic studies after stimulation with a potential agonist. Microplate assays have been described using ferricytochrome C, phenol red (Pick and Mizel,
1981), nitroblue tetrazolium (Pick, 1986) and scopoletin (De la Harpe and Nathan, 1985). The DCFH-DA based microassay offers the advantage over these test systems that fluorescence concentration increases with ongoing respiratory burst activity. However, the formation of oxidative products in those assays, is indicated by a decrease in a particular signal and is consequently associated with lower sensitivity. This applies both for scopoletin which after oxidation becomes nonfluorescent and indirectly also for the photometric measurement of ferricytochrome C or nitroblue tetrazolium since determination of absorbances insures measuring the diminution of light induced by a particular dye. Moreover, the differences in absorbance values in microplate assays using ferricytochrome C are often small sometimes making it difficult to clearly differentiate between stimulated cultures and controls (Pick and Mizel, 1981). Furthermore, unlike the scopoletin assay which requires added peroxidase, possible interference from reducing co-substrates is minimized in the DCFH-DA test. In conclusion, we have adapted a fluorometric burst assay for use in microtiter plates which permits reproducible detection of extracellular reactive oxygen intermediates. This technique should help to further elucidate the mechanisms of the respiratory burst response providing the facility for large scale evaluation of the role of surface receptors on phagocytes as well as for testing the effect of pro- and antiinflammatory substances. Since strong evidence exists for the extracellular release of superoxide anions and its dismutation product hydrogen peroxide in inflammatory processes such as the adult respiratory distress syndrome (Malech and Gallin, 1987), this assay, determining extracellular reactive oxygen intermediates, may provide a valuable addition to existing methods which assess intracellular burst formation.
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