Modfit lt cell cycle analysis software

Human nonexpanded and expanded PB-CD34⁺ cells (n = 5) were analyzed by flow cytometry (FACSCalibur flow cytometer, (Becton-Dickinson (BD), San Jose, CA). Dead cells were excluded by propidium iodide (PI) staining (Sigma, St Louis, MO). The CD34⁺ cells were incubated with a FcR blocking reagent (Miltenyi Biotec, Auburn, CA) and incubated with the monoclonal antibodies for 30 minutes at 4 °C. The stained cells were washed, resuspended, and then analyzed using Quad Statistics of CellQuest software (BD). The following monoclonal antihuman antibodies were used to characterized the CD34⁺ cell population: CD34-FITC (BD), CD31-PE (BD), CD133-PE (Miltenyi Biotec, Auburn, CA), CD68-PE (BD), CD83-PE (BD), VE-cadherin-PE (BD), VEGFR-2-PE (R&D Systems, Minneapolis, MN), Tie-2 (BD), CD117-PE (BD), CD45-PE (BD), IgG2a-FITC isotope controls (Miltenyi Biotec), and IgG1-PE isotope controls (Miltenyi Biotec).^{27 (link)}The DNA content analysis was assessed by staining ethanol-fixed cells with PI and monitoring with the FACSCalibur flow cytometer. At least 20,000 cells were collected and analyzed with CellQuest software. Cell cycle distributions were calculated with ModFit LT cell-cycle analysis software (Verity Software House, Topsham, ME).

Cell viability was determined by Trypan blue exclusion in a Countess automated cell counter (Invitrogen, Carlsbad, CA, USA). To evaluate cell cycle distribution, cells were harvested and processed as previously described.^{12 (link)} Samples were analyzed by flow cytometry (FACS Calibur; Becton-Dickinson, Franklin Lakes, NJ, USA) and cell cycle profiles were analyzed with ModFit LT Cell Cycle Analysis software (version 2.0) (Verity Software House, Topsham, ME, USA). Apoptosis was evaluated by staining cells with annexin V and propidium iodide (PI; Sigma-Aldrich), as previously detailed,^{12 (link)} and analyzed by flow cytometry. At least 50 000 events were acquired; data were processed with CellQuestPro software (Becton-Dickinson), and analyzed by Kaluza Analyzing Software v1.2 (Beckman Coulter, Pasadena, CA, USA). Annexin V-positive/PI-negative and annexin V-positive/PI-positive samples were classified as early and late apoptotic cells, respectively; both fractions were considered apoptotic cells. The percentage of specific cell death was estimated with the following formula: % cell death=100 x (percentage of dead cells in treated sample – percentage of dead cells in control)/(100% – percentage dead cells in control).

Cell lines were treated with AG1478 (at near IC₅₀ concentration) for 48 h. Cells were then collected by trypsinization and fixed with 70% ethanol by incubating them overnight at 4°C in the dark. The cell pellets were then resuspended in PBS and stained with propidium iodide/RNase staining buffer (BD Biociences) for 30 min at 37°C. Analysis was performed using a FACS Aria flow cytometer (Becton Dickenson, Franklin Lakes, NJ, USA), and the cell cycle data were processed using the ModFit LT cell cycle analysis software (Verity Software House, Topsham, ME, USA).