Isoluminol-enhanced chemiluminescence: A sensitive method to study the release of superoxide anion from human neutrophils

Surface-related triggering of the neutrophil respiratory burst. Characterization of the response induced by IgG adsorbed to hydrophilic and hydrophobic glass surfaces

“…Some of the IgG-coated tubes were further incubated in 5% human serum at room temperature for 30 min. A very sensitive chemiluminescence (CL) technique (well suited for real-time studies) was used for the determination of respiratory burst activity [26]. The glass tubes were filled with 1 ml of reaction mixture containing KRG, neutrophils (1 × 10 6 /ml), and luminol (5 × 10 ¹5 M), after which they were placed in disposable 4-ml polypropylene tubes and transferred into a six-channel Biolumat LB 9505 (Berthold Co., Wildbad, Germany).…”

Phorbol myristate acetate induces neutrophil NADPH-oxidase activity by two separate signal transduction pathways: dependent or independent of phosphatidylinositol 3-kinase

“…The CL method allows for the determination of both extracellularly released as well as intracellularly retained superoxide anion, exploiting the differences in membrane permeability between luminol (membrane permeable) and isoluminol (membrane impermeable) [28]. When stimulating the neutrophils with PMA, both intra-and extracellular CL was detected, indicating that the phorbol ester induces assembly of the NADPH-oxidase both in the plasma membrane, resulting in release of superoxide to the extracellular compartment, and in an intracellular membrane (probably the granule membrane), resulting in intracellular production of superoxide (Fig.…”

“…To quantify intracellularly and extracellularly generated superoxide, respectively, two different reaction mixtures were used. Tubes used for measurement of extracellular release of superoxide contained neutrophils, horseradish peroxidase (HRP; a cell-impermeable peroxidase; 4 units), and isoluminol (a cell-impermeable CL substrate; 2 ϫ 10 -5 M) [28]. Tubes used for measurement of intracellular generation of superoxide contained neutrophils, superoxide dismutase (SOD; a cell-impermeable scavenger for O 2 Ϫ ; 50 units), catalase (a cell-impermeable scavenger for H 2 O 2 ; 2000 units), and luminol (a cell-permeable CL substrate; 2 ϫ 10 -5 M).…”

Simultaneous use of electrochemistry and chemiluminescence to detect reactive oxygen species produced by human neutrophils

“…When using HRP as a catalyst, O 2 •-is the primary extracellular oxygen species in the peroxidasecatalysed excitation of LumH 2 , and thus H 2 O 2 released from the cells does not significantly affect the CL Lock et al, 1988;Lundqvist and Dahlgren, 1996).…”

“…However, placing the biosensors in a compartment with a spectral system (Chang et al, 2005b) requiring a dye probe to produce the optical signal, limits the applicability. Therefore, we performed the electrochemical and optical assays in two separate compartments along with simultaneous real- (Chang et al, 2005a;O'Neill et al, 2004) and by using more specific and/or sensitive CL compounds, like isoluminol which is highly selective with respect to extracellular ROS only Lundqvist and Dahlgren, 1996). Hence, it would be possible to extend our approach for simultaneous, direct, quantitative real-time measurements of ROS (e.g., O 2 •-and H 2 O 2 ), nitrogenous species (e.g., nitric oxide), and other messengers (e.g., glutamate)…”