Computational protocol: NADPH oxidase gp91phox contributes to RANKL-induced osteoclast differentiation by upregulating NFATc1

Similar protocols

Protocol publication

[…] Superoxide production was measured by an isoluminol chemiluminescence assay in 96-well plates using a SpectraMax L microplate reader (Molecular Devices). Osteoclast precursors (1 × 105) in PBSG (PBS with 0.9 mM CaCl2, 0.5 mM MgCl2, and 7.5 mM glucose) were added to each well of a 96-well plate with 125 μM isoluminol (Sigma) in 80 μL PBSG and 100 units HRP (Sigma) in 40 μL PBSG. After cells were incubated at 37 °C for 10 min, either 200 ng/mL PMA or 50 ng/mL RANKL was then injected into the wells by the automatic injector of the microplate reader. Chemiluminescence was detected as relative luminescence units by fast kinetic mode, and the amount of superoxide produced was determined using SoftMax PRO software. ROS levels in RANKL-treated osteoclasts were detected with 2′,7′-dichlorofluorescein diacetate (DCF-DA) as described previously. DCF, a highly fluorescent compound formed from DCF-DA as a result of exposure to ROS, was detected by fluorescence spectroscopy with maximum excitation and emission spectra of 495 nm and 529 nm, respectively. The DCF-DA stained cells were also examined under a fluorescence microscope (Carl Zeiss, Oberkochen, Germany). [...] Two-tailed Student’s t-test (paired) or one-way ANOVA for were performed using SigmaPlot11 software (Systat Software). Results were expressed as mean ± SD, and p values less than 0.05 were considered statistically significant. […]

Pipeline specifications

Software tools SoftMax Pro, SigmaPlot
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Mus musculus
Chemicals Hydrogen Peroxide, NADP, Oxygen, Superoxides