Diogenes

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- 75,000 cells/well were platted for the assay	- After 48h of post transfection, cells were for ROS with or without activation with 2 μg/ml phorbol myristate acetate

Upstream tips
- 75,000 cells/well were platted for the assay
Protocol tips
- After 48h of post transfection, cells were for ROS with or without activation with 2 μg/ml phorbol myristate acetate

Publication protocol

Cells were seeded in six-well dishes at 250,000 cells/well (HEK293 cells), 75,000 cells/well (CHO-K1 cells), or 700,000 cells/well (HT-29 cells) for 48 h prior to transfection. Cell transfections were performed in serum-free medium using 6 μl of FuGENE 6 prepared in complexes with plasmid DNAs (HEK293 and CHO-K1 cells, total, 2 μg/well; 0.5 μg of Nox1 or Nox3, plus 0.5 μg each of other expression vectors or pcDNA3.1 [control] plasmid; HT-29 cells, 0.5 μg each of expression vectors or pcDNA3.1 [control] plasmid without Nox1), using the manufacturer's suggested protocol; in studies expressing mutant products, equal moles of plasmid were used in place of wild type. The cells were fed 5 h posttransfection with complete medium and were assayed 48 h after transfection. Trypsinized cells were assayed for ROS release (with or without activation with 2 μg/ml phorbol myristate acetate [PMA]) by superoxide dismutase-sensitive chemiluminescence methods using the Diogenes reagent (National Diagnostics), as described previously (21). The reagent has 10,000 times greater sensitivity for superoxide than does hydrogen peroxide (J. Kitzler [National Diagnostics], personal communication).

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Manufacturer protocol

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