Synergy 4 hybrid multi mode microplate reader

The measurement of extracellular H₂O₂ production was performed using BMDMs and bone marrow neutrophils using a modified Amplex red assay. Briefly, H₂O₂ production was quantified in whole cells by Amplex^® Red as previously described^{28 –30 (link)}. Either bone marrow derived macrophages or bone marrow derived neutrophils were prepared as described above, resuspended in OptiMEM and then plated (4 × 10⁴ cells/well) into 96-well plate in assay buffer (25 mM Hepes, pH 7.4, containing 0.12 M NaCl, 3 mM KCl, 1 mM MgCl₂) supplemented with 0.1 mM Amplex^® Red, and 0.32 U/ml of horse radish peroxidase (HRP). The reaction was started by addition of 5μM phorbol 12-myristate 13-acetate (PMA). Fluorescence measurements were made using a Biotek Synergy 4 Hybrid Multi-Mode Microplate Reader with a 530/25 excitation and a 590/35 emission filter. The reaction was monitored at 37 °C for 1 h. A standard curve of known H₂O2 concentrations was included on each plate. NADPH oxidase (NOX) activity was obtained by calculating the rate of H₂O₂ production as RFU/min/40,000 cells after subtracting the equivalent value given by cells treated with 300 U/ml of catalase. Data are expressed as fold change of WT vehicle control.

Whole kidney tissue was homogenized in ice-cold phosphate buffer (PBS) and scraped in lysis buffer (8 mM potassium, sodium phosphate buffer pH 7.0, 131 mM NaCl, 340 mM sucrose, 2 mM NaN3, 5 mM MgCl2, 1 mM EGTA, 1 mM EDTA and protease inhibitors [Roche Diagnostics GmbH, Mannheim, Germany]). Tissue was further lysed by five freeze/thaw cycles, and passage through a 30-gauge (G) needle 5 times. The lysate was centrifuged at 1000 g (5 min; 4 °C) to remove unbroken cells, nuclei and debris. Extreme care was taken to maintain the lysate at a temperature close to 0 °C. The cell lysate was centrifuged at 28,000 g (15 min; 4 °C). The supernatant was removed, membranes were resuspended in lysis buffer, and protein concentration was measured using the Bradford microplate method.
Superoxide production in particulate fractions (20 μg/ml) of untreated, αCD47Ab-, rTM-, or sCR1-treated mice was measured in 0.1 ml of oxidase assay buffer (65 mM sodium phosphate buffer pH 7.0, 1 mM EGTA, 10 μM FAD, 1 mM MgCl2, 2 mM NaN3 and 0.2 mM cytochrome c [Sigma-Aldrich]). Superoxide production was initiated by the addition of 180 μM NADPH and was calculated from the initial linear rate of superoxide dismutase (SOD) (150 U/ml) (Sigma-Aldrich) inhibitable cytochrome c reduction quantified at 550 nm using an extinction coefficient of 21.1 mM-1 cm-1 (Biotek Synergy 4 Hybrid Multi-Mode Microplate Reader).