[59]
K I N E T I C M I C R O P L A T E ASSAY FOR 0 2 -
567
[59] K i n e t i c M i c r o p l a t e A s s a y for S u p e r o x i d e P r o d u c t i o n b y N e u t r o p h i l s a n d O t h e r P h a g o c y t i c Cells
By LAURA A. MAYO and JOHN T. CURNUTTE Introduction Phagocytic cells recognize and respond to a remarkable variety of stimuli which include opsonized microorganisms, chemotactic peptides, immune complexes, protein kinase C activators, membrane perturbants, and unsaturated fatty acids. One of the most dramatic responses of phagocytic cells to these types of stimuli is the respiratory burst, in which the rate of oxygen consumption abruptly increases by a factor of 10 to over 100. ~This oxygen participates in a nonmitochondrial reaction in which it is reduced by one electron to O2- by a plasma membrane-bound NADPH oxidase according to reaction (1). ~ Superoxide then serves as the preNADPH + 2 O2"-~ NADP + + H ÷ + 2 02-
(1)
cursor for a series of lethal oxidants such a s H 2 0 2 , HO', and HOCk Because superoxide is the initial product formed by NADPH oxidase and a substantial portion of it is released extracellularly, its measurement serves as a sensitive and convenient way of monitoring respiratory burst activity in phagocytic cells. The most widely used assay for monitoring O2- is the cytochrome c assay in which O2- reduces ferric cytochrome c to its ferrous form, a change which can be sensitively monitored at 550 nm2-4: Oxidized cytochrome c (Fe 3+) + 02- ~ reduced cytochrome c (Fe 2+) + O2
(2)
In complex cellular reaction mixtures, the extent to which O2- is responsible for the cytochrome c reduction can be determined by measuring the amount of this reduction that is sensitive to superoxide dismutase (SOD).2-4 While the activity of the respiratory burst can be monitored by a variety of assays for either O2- or other products of the burst, none offers the combined simplicity, specificity, and sensitivity of the cytochrome c assay when performed in the absence and presence of SOD.
i j. T. Curnutte 2 j. M. McCord 3 B. M. Babior, 4 j. T. Curnutte
and B. M. Babior, Adv. Hum. Genet. 16, 229 (1987). and I. Fridovich, J. Biol. Chem. 244, 6049 (1969). R. S. Kipnes, and J. T. Curnutte, J. Clin. Invest. 52, 741 (1973). and B. M. Babior, J. Clin. Invest. 53, 1662 (1974).
METHODS IN ENZYMOLOGY, VOL. 186
Copyright © 1990by Academic Press, Inc. All rights of reproduction in any form reserved.
568
LEUKOCYTES AND MACROPHAGES
[59]
A variety of assay procedures for measuring 02- production by phagocytic cells have been described in previous volumes of this series. 5-w Rather than reviewing or describing modifications of these assays, this chapter focuses on the adaptation of the cytochrome c assay to recently available kinetic microplate readers. Instrumentation
Superoxide production by phagocytic cells is usually monitored by either continuous or end-point assays in which cytochrome c reduction is measured with single- or dual-beam spectrophotometers. The continuous assay is the preferred method since the generation of 02- by phagocytes is not linear with respect to time. Depending on the stimulus, there may be a lag of between 5 sec and 10 min before the rate of production of 02reaches its maximal velocity. Arachidonic acid and the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine(FMLP), for example, have lag times of only several seconds, whereas fluoride anion does not cause 02- production for nearly 10 min. The duration of 02- production also varies considerably depending on the stimulus and is yet another reason why the continuous assay is preferable. In principle, either the continuous or end-point assay should be readily adaptable to 96-well microplates by simply decreasing proportionately the volumes of each of the reaction constituents and then measuring the absorbance change in a 96-well microplate photometer. An end-point assay for measuring 02- was developed along these lines and was described in this series by Pick in 1986/° Since that time, the development of microplate photometers capable of reading all 96 wells every 5 to 12 sec has made it possible to follow the kinetics of a wide variety of colorimetric assays in microplates. In developing a kinetic microplate assay for phagocyte O2- production, a number of technical problems had to be addressed, some of which were unique to the kinetic method. First, the temperature of the reaction wells has to be tightly controlled in order to optimize 02- production and minimize well-to-weU variability arising from inhomogeneities in temperature. Therefore, the microplate reader has to fit comfortably in a 37° incubator or have a thermostatted sample compartment. To further mini5 M. A. Trush, M. E. Wilson, and K. Van Dyke, this series, Vol 57, p. 462. 6 M. Markert, P. C. Andrews, and B. M. Babior, this series, Vol. 105, p. 358. 7 R. B. Johnston, Jr., this series, Vol. 105, p. 365. 8 p. j. O'Brien, this series, Vol. 105, p. 370. D. P. Clifford and J. E. Repine, this series, Vol. 105, p. 393. 10 E. Pick, this series, Vol. 132, p. 407.
[59]
KINETIC MICROPLATEASSAY FOR 02-
569
mize temperature variability, it is preferable that the photometer light source be located within the instrument in such a position that it does not cause additional heating of the sample chamber, Second, turbidity caused by the presence of intact cells in each reaction mixture decreases the light signal that reaches the detector. Therefore, the light source must have sufficient intensity to penetrate reaction mixtures containing relatively high concentrations of cells. A third problem, also related to the presence of intact cells in each reaction, is that substantial light scatter can occur and result in "cross-talk" between wells. Fourth, in all but the briefest incubations, the reaction wells must be agitated regularly to keep the cells in a homogeneous suspension and thereby optimize the reduction of cytochrome c by 02-. Fifth, the light path in each reaction mixture has to be uniform and not vary during the course of the incubation. This is best accomplished using microplates made of hydrophilic plastic in which the meniscus is controlled by gravity and is therefore uniform in each well. Sixth, the absorption peak at 550 nm of the reduced minus oxidized cytochrome c spectrum is relatively narrow, necessitating the use of a narrow bandwidth optical filter. In light of the above considerations, the microplate reader chosen for the development of the kinetic 02- assay was the Vmax kinetic reader manufactured by Molecular Devices (Menlo Park, CA). Other commercially available kinetic microplate readers were not tested in this study. The Vmax was used in a custom-built plexiglass 37° incubator equipped with a front access door. The instrument performed well under these conditions. (The manufacturer of the Vmax can be contacted for information regarding a recently available thermostatted microplate reader.) The reading chamber in the Vmax has a temperature variation of less than -+1.0% under these conditions, owing in part to the location of the lowwattage light source away from the sample chamber. Individual wells are sequentially illuminated by an array of fiber optic channels. Besides contributing to the temperature stability of the samples, this fiber optic arrangement allows 100% of the available light to be delivered to each well independently, thereby improving the accuracy and linearity of the absorbance measurements, particularly with turbid samples. A focal lens array serves to focus the light transmitted through each well onto its own detector. The microplate carriage is capable of agitation both before and between readings, still allowing measurements to be performed every 8 sec. Uniformity of each light path was achieved using constant reaction volumes in hydrophilic plates (Nunclon, Vangard, Inc., Neptune, N J). The Vmax microplate reader was equipped with separate 550-nm optical filters with bandwidths of either 10 or 1 nm. Figure 1 shows the absorbance difference between dithionite-reduced and oxidized cyto-
570
LEUKOCYTES AND MACROPHAGES i
i
|
J
1.2
[59] !
*
i
~p~/•
Q
o
tO
0.8
0
0 m
.a
K I N E T I C M I C R O P L A T E ASSAY FOR 0 2 -
567
[59] K i n e t i c M i c r o p l a t e A s s a y for S u p e r o x i d e P r o d u c t i o n b y N e u t r o p h i l s a n d O t h e r P h a g o c y t i c Cells
By LAURA A. MAYO and JOHN T. CURNUTTE Introduction Phagocytic cells recognize and respond to a remarkable variety of stimuli which include opsonized microorganisms, chemotactic peptides, immune complexes, protein kinase C activators, membrane perturbants, and unsaturated fatty acids. One of the most dramatic responses of phagocytic cells to these types of stimuli is the respiratory burst, in which the rate of oxygen consumption abruptly increases by a factor of 10 to over 100. ~This oxygen participates in a nonmitochondrial reaction in which it is reduced by one electron to O2- by a plasma membrane-bound NADPH oxidase according to reaction (1). ~ Superoxide then serves as the preNADPH + 2 O2"-~ NADP + + H ÷ + 2 02-
(1)
cursor for a series of lethal oxidants such a s H 2 0 2 , HO', and HOCk Because superoxide is the initial product formed by NADPH oxidase and a substantial portion of it is released extracellularly, its measurement serves as a sensitive and convenient way of monitoring respiratory burst activity in phagocytic cells. The most widely used assay for monitoring O2- is the cytochrome c assay in which O2- reduces ferric cytochrome c to its ferrous form, a change which can be sensitively monitored at 550 nm2-4: Oxidized cytochrome c (Fe 3+) + 02- ~ reduced cytochrome c (Fe 2+) + O2
(2)
In complex cellular reaction mixtures, the extent to which O2- is responsible for the cytochrome c reduction can be determined by measuring the amount of this reduction that is sensitive to superoxide dismutase (SOD).2-4 While the activity of the respiratory burst can be monitored by a variety of assays for either O2- or other products of the burst, none offers the combined simplicity, specificity, and sensitivity of the cytochrome c assay when performed in the absence and presence of SOD.
i j. T. Curnutte 2 j. M. McCord 3 B. M. Babior, 4 j. T. Curnutte
and B. M. Babior, Adv. Hum. Genet. 16, 229 (1987). and I. Fridovich, J. Biol. Chem. 244, 6049 (1969). R. S. Kipnes, and J. T. Curnutte, J. Clin. Invest. 52, 741 (1973). and B. M. Babior, J. Clin. Invest. 53, 1662 (1974).
METHODS IN ENZYMOLOGY, VOL. 186
Copyright © 1990by Academic Press, Inc. All rights of reproduction in any form reserved.
568
LEUKOCYTES AND MACROPHAGES
[59]
A variety of assay procedures for measuring 02- production by phagocytic cells have been described in previous volumes of this series. 5-w Rather than reviewing or describing modifications of these assays, this chapter focuses on the adaptation of the cytochrome c assay to recently available kinetic microplate readers. Instrumentation
Superoxide production by phagocytic cells is usually monitored by either continuous or end-point assays in which cytochrome c reduction is measured with single- or dual-beam spectrophotometers. The continuous assay is the preferred method since the generation of 02- by phagocytes is not linear with respect to time. Depending on the stimulus, there may be a lag of between 5 sec and 10 min before the rate of production of 02reaches its maximal velocity. Arachidonic acid and the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine(FMLP), for example, have lag times of only several seconds, whereas fluoride anion does not cause 02- production for nearly 10 min. The duration of 02- production also varies considerably depending on the stimulus and is yet another reason why the continuous assay is preferable. In principle, either the continuous or end-point assay should be readily adaptable to 96-well microplates by simply decreasing proportionately the volumes of each of the reaction constituents and then measuring the absorbance change in a 96-well microplate photometer. An end-point assay for measuring 02- was developed along these lines and was described in this series by Pick in 1986/° Since that time, the development of microplate photometers capable of reading all 96 wells every 5 to 12 sec has made it possible to follow the kinetics of a wide variety of colorimetric assays in microplates. In developing a kinetic microplate assay for phagocyte O2- production, a number of technical problems had to be addressed, some of which were unique to the kinetic method. First, the temperature of the reaction wells has to be tightly controlled in order to optimize 02- production and minimize well-to-weU variability arising from inhomogeneities in temperature. Therefore, the microplate reader has to fit comfortably in a 37° incubator or have a thermostatted sample compartment. To further mini5 M. A. Trush, M. E. Wilson, and K. Van Dyke, this series, Vol 57, p. 462. 6 M. Markert, P. C. Andrews, and B. M. Babior, this series, Vol. 105, p. 358. 7 R. B. Johnston, Jr., this series, Vol. 105, p. 365. 8 p. j. O'Brien, this series, Vol. 105, p. 370. D. P. Clifford and J. E. Repine, this series, Vol. 105, p. 393. 10 E. Pick, this series, Vol. 132, p. 407.
[59]
KINETIC MICROPLATEASSAY FOR 02-
569
mize temperature variability, it is preferable that the photometer light source be located within the instrument in such a position that it does not cause additional heating of the sample chamber, Second, turbidity caused by the presence of intact cells in each reaction mixture decreases the light signal that reaches the detector. Therefore, the light source must have sufficient intensity to penetrate reaction mixtures containing relatively high concentrations of cells. A third problem, also related to the presence of intact cells in each reaction, is that substantial light scatter can occur and result in "cross-talk" between wells. Fourth, in all but the briefest incubations, the reaction wells must be agitated regularly to keep the cells in a homogeneous suspension and thereby optimize the reduction of cytochrome c by 02-. Fifth, the light path in each reaction mixture has to be uniform and not vary during the course of the incubation. This is best accomplished using microplates made of hydrophilic plastic in which the meniscus is controlled by gravity and is therefore uniform in each well. Sixth, the absorption peak at 550 nm of the reduced minus oxidized cytochrome c spectrum is relatively narrow, necessitating the use of a narrow bandwidth optical filter. In light of the above considerations, the microplate reader chosen for the development of the kinetic 02- assay was the Vmax kinetic reader manufactured by Molecular Devices (Menlo Park, CA). Other commercially available kinetic microplate readers were not tested in this study. The Vmax was used in a custom-built plexiglass 37° incubator equipped with a front access door. The instrument performed well under these conditions. (The manufacturer of the Vmax can be contacted for information regarding a recently available thermostatted microplate reader.) The reading chamber in the Vmax has a temperature variation of less than -+1.0% under these conditions, owing in part to the location of the lowwattage light source away from the sample chamber. Individual wells are sequentially illuminated by an array of fiber optic channels. Besides contributing to the temperature stability of the samples, this fiber optic arrangement allows 100% of the available light to be delivered to each well independently, thereby improving the accuracy and linearity of the absorbance measurements, particularly with turbid samples. A focal lens array serves to focus the light transmitted through each well onto its own detector. The microplate carriage is capable of agitation both before and between readings, still allowing measurements to be performed every 8 sec. Uniformity of each light path was achieved using constant reaction volumes in hydrophilic plates (Nunclon, Vangard, Inc., Neptune, N J). The Vmax microplate reader was equipped with separate 550-nm optical filters with bandwidths of either 10 or 1 nm. Figure 1 shows the absorbance difference between dithionite-reduced and oxidized cyto-
570
LEUKOCYTES AND MACROPHAGES i
i
|
J
1.2
[59] !
*
i
~p~/•
Q
o
tO
0.8
0
0 m
.a
