Lv200 luminescence microscopy system

Precision-cut lung sections were prepared via vibratome (Integrasection 7550MM; Campden Instruments, Loughborough, United Kingdom) with a thickness of 275 μm (19 (link)). Sections were placed on cell culture inserts within 35-mm, glass-bottomed dishes containing 1 ml of luciferin recording medium, sealed by a glass coverslip. Photon count was recorded in 1 h time bins with an LV200 luminescence microscopy system (Olympus, Tokyo, Japan), as previously described by Gibbs et al. (3 (link)). Bronchiole and parenchyma regions were selected and analyzed with ImageJ software. Bioluminescence from macrophages was recorded by a photomultiplier tube system, as previously described by Gibbs et al. (20 (link)). Data was detrended with a 24-h moving average and plotted as relative bioluminescence (photons/min). Phase change was calculated as the timing of the fourth peak of treated samples relative to the fourth peak of untreated samples (19 (link)). To test responses to ligands of the Gc and mineralocorticoid receptor (MR), we used corticosterone (100 nM; MilliporeSigma), RU486 (mifepristone, 1 µM; MilliporeSigma), Dex (200 nM; MilliporeSigma), 2 synthetic nonsteroidal compounds [67 and 69 (21 (link))], used at 10 nM, a kind gift from Dr. Stuart Farrow, (GlaxoSmithKline, Brentford, United Kingdom), and an agonist of the MR spironolactone (1 µM; MilliporeSigma).

Precision-cut ectopic lung slices (275 μm) were prepared as previously described (39 (link)). After washes to remove residual agarose, slices were placed onto cell culture inserts (Millicell) within 35-mm dishes containing 1 ml recording medium and sealed with a coverslip. Dishes were then transferred to a 37°C incubator housing the photomultiplier tubes (PMTs; H6240 MOD1; Hamamatsu Photonics) or placed under a self-contained Olympus LV200 luminescence microscopy system fitted with a cooled Hamamatsu C9100-13 EM-CCD camera (Olympus) as previously described (40 (link)). Individual regions of interest were delineated using ImageJ software (version 1.41o; NIH).