Facscanto cytofluorometer

Apoptosis was measured by flow cytometry using an Annexin V-FITC Apoptosis Detection kit I (BD Pharmingen, San Diego, CA). Briefly, cells were cultured in MEM supplemented with 10% FBS, 1 × 10⁶ cells were washed with cold PBS and then resuspended in 1 × cold binding buffer plus 5 μl Annexin V-FITC and 5 μl PI (propidium iodide). After 15 min incubation at RT in the dark, samples were analyzed by dual color flow cytometry on a FACSCanto Cytofluorometer (BD Pharmingen, San Diego, CA).

Cells were seeded in 100 mm dishes at a density of 5 × 10⁵ per dish. Twenty-four hours later, medium was changed and after 48 hours both adherent and floating cells were harvested and lysed for 10 minutes in lysing buffer (0.1% sodium citrate) (Sigma, St Louis, Missouri, USA), 0.1% Triton X-100 (Sigma, St Louis, Missouri, USA), 40 μg/ml Propidium Iodide (Sigma, St Louis, Missouri, USA). Cells were analyzed for DNA content by flow cytometry with FACSCanto Cytofluorometer (BD Pharmingen, San Diego, CA) and the cell-cycle phases were analyzed using FCS Express V3 with MultiCycle AV (http://www.denovosoftware.com). The experiments were performed twice with similar results.