In Vivo Bioluminescence Imaging of NF-κB Activity during Reperfusion

Two days before bioluminescence imaging, mice were shaved dorsally at the sites of the kidneys to prevent any quenching of the bioluminescent signal by the fur. κ-Luc reporter mice were anesthetized with 2.5% isoflurane and injected subcutaneously with 1.5 mg d-luciferin (VivoGlo, Promega) in 100 μl PBS (pH, 7.0). Whole-body image was acquired after an exposure time of 60 seconds using an In Vivo Imaging System (IVIS Spectrum, PerkinElmer, Waltham, MA). Mice were measured at different time points after ischemia, as depicted in Figure 1A. To obtain the luciferin kinetics, every 2 minutes an image was acquired subsequently 10 times after d-luciferin injection. From the luciferin kinetics, the measurements showing maximal radiance were used for quantification. Using Living Image software, version 4.2 (Caliper LifeSciences), the bioluminescent signal was quantified. Data are presented in physical units of radiance in photons/sec per cm² per steradian.