It offers the advantage of testing cells online for their response to a number of stimuli (PMA, zymosan, serum-treated zymosan, PAF/fMLP) over a prolonged time-period. This is a distinct advantage when testing cells from CGD patients with hypomorphic mutations, such as X91− CGD patients, which show less NADPH oxidase activity than normal cells but distinctly more than cells from ‘classical’ CGD patients. For details, see Protocol 1. It should be kept in mind that the Amplex Red assay is not really a quantitative assay, as it overestimates low NADPH oxidase

activities. This may be due to catalase in the neutrophils screening assay more efficiently removing high than low levels of intracellularly formed H2O2 before it can be detected in the extracellular medium. An alternative assay for such patients is the ferricytochrome c assay (see section Superoxide production), which can also be used with various NADPH oxidase stimuli. NB: Control cells should also be tested! Materials: Microplate reader: Genios Plus, Tecan 96-well microtitre plates, flat-bottomed, white polystyrene: Costar Amplex Red: Molecular Probes, cat no. A-12212, HIF inhibitor 5 mg Horseradish peroxidase (HRP): Sigma, cat no. P-8250, 5000 U Zymosan: MP Biomedicals Serum-treated zymosan (STZ):

see Goldstein et al., J Clin Invest 1975; 56:1155–63 Phorbol myristate acetate (PMA): Sigma Formyl-methionyl-leucyl-phenylalanine Mirabegron (fMLP): Sigma Platelet-activating factor (PAF): Sigma Prepare 20 mM Amplex Red in dimethylsulphoxide (DMSO), aliquots of 12·5 μl in −20°C Prepare 200 U/ml HRP in phosphate-buffered saline (PBS), aliquots of 25 μl in −20°C Solutions: Prepare ×2 reaction mix: Add 1 ml of HEPES to the Amplex Red aliquot and 1 ml of HEPES to the HRP aliquot and transfer

to 3 ml of HEPES medium to make 5 ml of ×2 reaction mix Method: Open ‘Amplex Red’ mode on plate reader (Ex 535 nm, Em 590 nm, interval 30 s, 61 cycles, 2 s of shaking before and in between cycles, 37°C) Pipette (no air bubbles!!) in white 96-well plate (do not use outer wells) 100 μl of ×2 reaction mix 50 μl of cell suspension Place 96-well plate in plate reader, and click ‘plate in’ (preincubation at 37°C). Click after 5 min ‘plate out’ Pipette 50 μl of stimulus (no air bubbles!!) Click ‘Start’ directly (NB: reaction to fMLP is very quick and transient) Luminol is a ROS probe with chemiluminescent properties. It enters cells and therefore detects both intra- and extracellular H2O2. By adding SOD and catalase, to remove extracellular O2− and H2O2, the reaction can be made specific for intracellular ROS. The luminol assay relies, again, on the availability of intracellular peroxidase and thus again carries the danger of misdiagnosing MPO deficiency for CGD. Detailed protocols for this assay can be found in [14, 17].