An in-house collection of drug-like compounds with diverse chemical scaffolds was screened at a final concentration of 7 μM by using a high-throughput circadian assay system, as described previously (15, 16, 22). In follow-up studies, stable U2OS reporter cells harboring Bmal1-dLuc or Per2-dLuc reporters (34) were suspended in culture medium [Dulbecco’s modified Eagle medium (DMEM; Gibco, 11995-073) supplemented with 10% fetal bovine serum (FBS), l-glutamine (0.29 mg/ml), penicillin (100 U/ml), and streptomycin (100 μg/ml)] and plated onto a white, solid-bottom 384-well plate at 30 μl (3000 cells) per well. After 2 days, 40 μl of explant medium [DMEM (Gibco, 12800-017) supplemented with 2% B27 (Gibco), 10 mM Hepes, sodium bicarbonate (0.38 mg/ml), l-glutamine (0.29 mg/ml), penicillin (100 U/ml), streptomycin (100 μg/ml), and 0.2 or 1 mM luciferin (pH 7.2)] was dispensed into each well, followed by the application of 500 nl of compounds [dissolved in dimethyl sulfoxide (DMSO); final 0.7% DMSO]. The plate was covered with an optically clear film, and luminescence was recorded every 100 min for 5 days in a microplate reader [Infinite M200 PRO (Tecan) or Synergy2 (BioTek)].

Lung tissue was dissected from Per2::Luc knock-in mice (57). Spleen and bone marrow tissues were dissected from Per2::Luc MLL-AF9 leukemia mice and control transplant mice (see below). For leukemic mice, tissues were collected when mice exhibited typical symptoms due to leukemia (e.g., weakness and slow movements) at 3 to 4 weeks after transplantation with leukemic cells. The tissue pieces were cultured in explant medium containing compounds (final 0.24% DMSO) in a black, clear-bottom 24-well plate. The plate was covered with an optically clear film, and luminescence was recorded every 30 min for 5 days in a LumiCEC luminometer (Churitsu Electric Corporation).

Circadian period was determined from luminescence rhythms by a curve-fitting program, MultiCycle (Actimetrics). The luminescence intensity was calculated by averaging the intensity during the entire experiment. Data from the first day were excluded from analysis, because of transient changes in luminescence upon medium change. LogEC50 was obtained by sigmoidal dose-response fitting of dilution series data (threefold, 12 points) with Prism software (GraphPad Software).

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