Collagen 1 coated 12 well plate

Differentiated podocytes (12 × 10⁵ cells per well) were plated in a collagen-I-coated 12-well plate (BD Biosciences). Cells were then given various treatments (LPS, 100 ng/ml; poly(I:C), 300 ng/ml; arginine, 1 mM) for 18 h in phenol-free, nitrate/nitrite-free RPMI at 37 °C. The supernatant was collected and filtered. Readings for each sample (100 μl) were performed using a chemiluminescence detector (Model 280i; Sievers, Boulder, CO, USA), with vanadium chloride at 95 °C as a reducing agent as described [23 (link)]. Measurements were performed in triplicates and reported in micromolar concentrations.

Differentiated podocytes (2 × 10⁵ cells per well) were plated in a collagen-I-coated 12-well plate (BD Biosciences). The cells were then given various treatments (LPS, 100 ng/ml; poly(I:C), 300 ng/ml; arginine, 1 mM) for 18 h. The cells were homogenized in buffer (pH 7.4) with 150 mM NaCl, 50 mM Tris, 25 mM ethylene glycol tetraacetic acid, and 25 mM ethylenediaminetetraacetic acid with a protease inhibitor and phosphatase inhibitor cocktail (source). Freshly prepared homogenates (100 ml) were incubated for 30 min at 37 °C with no treatment, or with ascorbate as a negative control, or with one of the following inhibitors of known ${\mathrm{O}}_2^{•-}$ diphenylene -producing enzymes: diphenylene iodonium (50 mM) or 7-(1,3-benzoxazol-2-ylsulfanyl)-3-benzyl-3H-[1–3]triazolo[4,5]–d pyrimidine (VAS2870; 10 μM, Life Sciences). Incubated samples (50 μl) were placed in a 24-well reading plate containing 25 μM lucigenin (Sigma) for the detection of ${\mathrm{O}}_2^{•-}$ in 500 μl final volume of Krebs solution with 10 mM Hepes–NaOH (pH 7.4). Readings for each sample were performed using a Luminoskan luminometer (Thermo Scientific, Waltham, MA, USA) at 37 °C in triplicates and normalized to the protein level measured by protein assay (Bio-Rad, Hercules, CA, USA) after background subtraction.