The Amplex Red kit (Molecular Probes) was used to determine the amount of reactive oxygen species produced by NB4 control or reconstituted cells, as previously described (52 (link)). 2.5 × 105 cells/mL were incubated with Amplex Red (25 μM) and horseradish peroxidase (0.5 U/mL). 5 min later, cells were stimulated with 100 ng/mL phorbol 12-myrisatate 13-acetate (PMA; Sigma-Aldrich), 1 mg/mL unopsonized zymosan (MP Biomedicals), or serum-treated zymosan (STZ), for 30 min at 37°C. Every 30 s, the fluorescence was assessed with a GENios plate reader (Tecan). The maximal slope of H2O2 release was measured with a 2-min interval at an excitation wavelength of 535 nm and an emission wavelength of 595 nm.
Unopsonized zymosan
Manufactured by MP Biomedicals
Unopsonized zymosan is a cell wall extract derived from the Saccharomyces cerevisiae yeast. It is a complex carbohydrate that can be used to stimulate the innate immune response in various experimental settings.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using unopsonized zymosan
1
Quantifying Cellular ROS Production
2
Measuring Hydrogen Peroxide Release
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The release of hydrogen peroxide was measured by using the Amplex Red kit (Molecular probes), as described earlier (19) . Note that 0.25 Â 10 6 cells were stimulated with Escherichia coli (OD625 ¼ 0.2, strain ML-35; 0.25 Â 10 9 per mL) in the presence of 2 mmol/L azide, platelet-activation factor (PAF; 100 nmol/L; Sigma-Aldrich)/Nformylmethionine-leucyl-phenylalanine (fMLP; 30 nmol/L; Sigma Aldrich), phorbol 12-myristate 13-acetate (PMA; 100 ng/mL; Sigma Aldrich), serum-treated zymosan (21) , or unopsonized zymosan (1 mg/mL; MP Biomedicals) for 30 minutes at 37 C. Every 30 seconds, fluorescence was assessed with a Genios plate reader (Tecan). Maximal slope of H 2 O 2 release was measured at 2-minute intervals at an excitation wavelength of 535 nm and an emission wavelength of 595 nm. Analysis is shown as the maximal slope in relative fluorescence units/minute.
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