Modfit lt program

Cell cycle was determined by flow cytometric analysis after propidium iodide (PI) staining. In brief, the suspension (2×10⁶ cells/ml) of 3T3-L1 pre-adipocytes were divided into 6 well culture plates (2 ml/well) and incubated for 48 h in DMEM containing 10% FBS. After that, the media were switched to a serum-free DMEM and further incubated for 12 h to induce a mild synchronization. The cells were then exposed to various PET-LM concentrations (0–100 μg /ml) and cultured for additional-24 h without any treatment with FBS or adipogenic differentiating factors. Cells were harvested by a trypsinization method and resuspended in PBS containing 25% ethanol followed by incubation at −20°C for overnight. After removing the fixation solution, cells were incubated at room temperature for 30 min in a mixture containing 50 μg/ml PI and 500 units/ml RNase. The samples were stored in the dark at 4°C before cell cycle progression analysis and 10,000 cells in each experiment were counted to measure the PI intensity using a FACS Vantage® System (Becton Dickinson, San Jose, CA, USA). The cell cycle progression was determined using the ModFit LT program (Verity Software House, Topsham, ME, USA).

Cells cultured in 6-well plates were harvested, washed once in PBS and fixed in 70 % ethanol for 48 h at 4 °C. The nuclei were stained with 50 μg/ml propidium iodide (PI) in 1 % Triton-X100/PBS containing 100 μg/ml DNase-free RNase, and the DNA content was analyzed using flow cytometry with the FACSCalibur platform (Becton Dickinson, San Jose, USA). The proportion of cells in each phase of the cell cycle was determined using the ModFit LT program (Verity Software House, USA).

Cells were detached using 0.1% trypsin in 2.5 mM EDTA and washed with ice-cold PBS. Subsequently, the cells were fixed with 66% (v/v) ethanol on ice for a least 30 min and washed with ice-cold PBS. Next, the cells were digested using RNase A (500 U/mL) for 30 min at 37 °C and stained with propidium iodide (50 µg/mL). The DNA content (10,000 cells per experimental group) was determined using a FACS Calibur flow cytometer (Becton Dickinson Biosciences, San Jose, CA, USA) equipped with a ModFit LT program (Verity Software House, Topsham, ME, USA).

Analyses of Fas/CD95 surface expression, cleaved Caspase-8, and measurement of mitochondrial membrane potential were performed as previously described [6 (link)]. The anti-Fas-PE (CD95; 555674) and anti-TRAIL-PE (CD-253; 550516) antibodies were purchased from BD Biosciences (Milan, Italy).
For cell cycle analyses, 6×105 proliferating and senescent cells were seeded into 60mm dishes. After 24h, cells were incubated with 10 μM 5-bromo-2-deoxyuridine (BrdU) for 30 min (proliferating) or 1h (senescent), fixed with ethanol and routinely kept at -20°C overnight. After two washes with phosphate-buffered saline (PBS), cells were incubated in 4M HCl for 30 min. Cells were washed with 0.1% Tween 20 in PBS, and incubated with FITC-conjugated anti-BrdU antibody (BD Biosciences, San Jose, CA, USA), according to the instructions of the manufacturer. Cells were washed with 0.1% Tween 20 in PBS, treated with 50μg/ml RNase DNase-free and 50μg/ml propidium iodide for 20 min, and analyzed using an Accuri C6 flow cytometer (BD Bioscience). Cell cycle distribution was analyzed by ModFit LT program (Verity Software House Inc., USA).