O2•− producing MDRC were detected by flow cytometry after staining with myeloid cell-specific antibodies and incubation for 20 min at RT with dihydroxyethidium (DHE, 10 μM; Molecular Probes, Eugene, OR) as described before 32 (link). The specificity of DHE for O2•− was validated by inducing a respiratory burst in the sorted myeloid cells by incubation at 37°C for 15 min with phorbol myristate acetate (PMA, 1 μg/ml) or PMA + the NADPH oxidase inhibitor diphenylene iodonium (DPI, 1 μM; Tocris Bioscience, Bristol, UK) , in presence of superoxide dismutase (256mU/ml, Sigma, St. Louis, MO) or in presence or absence of 10μM Pyrrolidine dithiocarbamate (Calbiochem, La Jolla, CA) or in the presence or absence of 5 μM NF- κB activation inhibitor II, JSH-23 ( Santa Cruz Biotechnology, Santa Cruz, CA ).
Protocol detail
Myeloid cell superoxide detection
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Antibodies
Cells respiratory
Diphenylene iodonium
Flow cytometry
Jsh 23
Molecular probes
Myeloid cell
Nadph oxidase
Nf κb inhibitor
Pyrrolidine dithiocarbamate
Superoxide dismutase
Corresponding Organization : University of Alabama at Birmingham
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Variable analysis
independent variables
- Incubation with phorbol myristate acetate (PMA, 1 μg/ml)
- Incubation with PMA + the NADPH oxidase inhibitor diphenylene iodonium (DPI, 1 μM)
- Presence of superoxide dismutase (256mU/ml)
- Presence or absence of 10μM Pyrrolidine dithiocarbamate
- Presence or absence of 5 μM NF-κB activation inhibitor II, JSH-23
- O2•− production detected by flow cytometry after staining with myeloid cell-specific antibodies and incubation with dihydroxyethidium (DHE, 10 μM)
- Incubation time of 20 min at room temperature (RT) with dihydroxyethidium (DHE, 10 μM)
- Induction of respiratory burst in sorted myeloid cells by incubation at 37°C for 15 min with phorbol myristate acetate (PMA, 1 μg/ml)
- Incubation with PMA + the NADPH oxidase inhibitor diphenylene iodonium (DPI, 1 μM)
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