Cellular dna flow cytometric analysis kit

Cell cycle analysis was performed, as described previously [7 (link)]. Briefly, Rh30 or MDA-MB-231 cells were treated with CPX at 5 μM for 0-72 h, followed by staining with the Cellular DNA Flow Cytometric Analysis Kit (Roche Diagnostics, Indianapolis, IN, USA). Percentages of cells within each of the cell cycle compartments (G₀/G₁, S, or G₂/M) were determined using a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA, USA) and ModFit LT analyzing software (Verity Software House, Topsham, ME, USA). Cells treated with vehicle alone (100% ethanol) were used as a control.

Cells were seeded in 100-mm dishes at a density of 5×10⁵ cells per dish in DMEM supplemented with 10% FBS and grown overnight at 37°C in a humidified incubator with 5% CO₂. The next day, cells were then treated with FC101 at 0–5 µM for 24 h or at 1 µM for 0–72 h. Subsequently, the cells were briefly washed with PBS and trypsinized. Cell suspensions were centrifuged at 1,000 rpm for 5 min, and the pellets were fixed with pre-chilled (−20°C) absolute ethanol and stained with the Cellular DNA Flow Cytometric Analysis Kit (Roche Diagnostics Corp., Indianapolis, IN). Percentages of cells within each of the cell cycle compartments (G₀/G₁, S, or G₂/M) were determined using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA) and ModFit LT analyzing software (Verity Software House, Topsham, ME). Cells treated with vehicle alone (PBS) served as a control. Also, the confluence of the control cells was kept <80%, to minimize a possible effect of the cell-cell contact inhibition on cell cycle progression.

Maduramicin ammonium (molecular weight = 934.16, purity>97%, by HPLC) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution (5 mg/ml), aliquoted and stored at −80°C. Dulbecco's modified Eagle's medium (DMEM) and 0.05% trypsin-EDTA were obtained from Mediatech (Manassas, VA, USA). Fetal bovine serum (FBS) was from Atlanta Biologicals (Lawrenceville, GA, USA). One Solution Cell Proliferation Assay Kit was from Promega (Madison, WI). Cellular DNA Flow Cytometric Analysis Kit was purchased from Roche Diagnostics (Indianapolis, IN, USA). CF488A-Annexin V and Propidium Iodide (PI) Apoptosis Assay Kit was purchased from Biotium (Hayward, CA, USA). Enhanced chemiluminescence solution was from Perkin-Elmer Life Science (Boston, MA, USA). The following antibodies were used: cyclin A, cyclin B1, cyclin D1, cyclin E, CDK1, CDK2, CDK4, CDK6, CDC25A, CDC25B, CDC25C, p21^Cip1, p27^Kip1, Rb, p-Rb (S807/811), survivin, Mcl-1, Bcl-2, Bcl-xL, BAX, BAK, BAD, FasL, Fas/CD95, TNFα, TNFR1, TRAIL, DR4, DR5, FLIP S/L, FADD, TRADD, RIP (Santa Cruz Biotechnology, Santa Cruz, CA, USA), cleaved caspase 3, cleaved PARP (Cell Signaling, Beverly, MA, USA), β-tubulin (Sigma, St Louis, MO), goat anti-mouse IgG-horseradish peroxidase, and goat anti-rabbit IgG-horseradish peroxidase (Pierce, Rockford, IL, USA).