Mouse antiserum

raised against α−tubulin was purchased by Calbiochem (Merck KGaA, Darmstadt, Germany). 5-FU, Doxorubicin and were Levofolene were a gift of Dr. Gaetano Facchini (I.N.T. ‘Pascale’, Naples, Italy). Cell culture and proliferation The rat cardiocytes (H9c2) cell line and the human colon adenocarcinoma (HT-29) cell line obtained from the American Type Tissue Culture Collection, Rockville, MD, grow in DMEM and RPMI1640, respectively, selleck chemicals supplemented with heat inactivated 20% FBS, 20 mM HEPES, 100 U/ml penicillin, 100 mg/ml streptomycin, 1% L-glutamine and 1% sodium pyruvate. Both cell lines were grown in a humidified atmosphere of 95% air/5% CO2 at 37 °C. Proliferation of H9c2 and HT-29 cell lines was performed in the presence of 5-FU and Doxorubicin (DOXO) in presence or not of Levofolene (LF), by MTT assay as previously described [28]. Western blot analysis H9c2 and HT-29 cell lines were grown for 48 h with or without DOXO or 5-FU in presence MK-4827 cost or not of LF at 37°C. For cell extract preparation, the cells were washed twice with ice-cold PBS, scraped and centrifuged for 30 min at 4°C in 1 ml of lysis buffer (1% Triton, 0.5% sodium deoxycholate, 0.1 NaCl, 1 mM EDTA, pH 7.5, 10 mM Na2HPO4, pH 7.4, 10 mM PMSF, 25 mM benzamidin, 1 mM leupeptin, 0.025 units/ml aprotinin). Equal amounts of cell proteins

were separated by SDS-PAGE, electrotransferred to nitrocellulose and reacted with the different antibodies. Blot were then developed using enhanced chemiluminescence detection reagents (SuperSignal West Pico, Pierce) and exposed to x-ray film. All films were scanned by using Quantity One software (BioRad laboratories, Hercules, CA). Flow cytometric analysis of apoptosis Annexin CB-5083 mouse V-FITC (fluorescein isothiocyanate) was used in conjunction with a vital dye, Propidium Iodide (PI), to distinguish apoptotic (Annexin V-FITC positive, PI negative) from necrotic (Annexin V-FITC positive, propidium iodide positive) cells. Briefly, cells were incubated with Annexin-V–FITC (MedSystems Diagnostics, Vienna, Austria) and propidium iodide (Sigma, St. Louis, MO, USA) in a binding buffer (10 mM Hepes, pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM

MgCl2, 2.5 mM CaCl2) for 10 min at room temperature, washed and resuspended in Thalidomide the same buffer. Analysis of apoptotic cells was performed by flow cytometry (FACScan, Becton Dickinson). For each sample, 2 × 104 events were acquired. Analysis was carried out by triplicate determination on at least three separate experiments. Flow cytometric analysis of oxidative stress The cells were seeded in 6-multiwell plates at the density of 3 × 105 cells/well. After 24 h incubation at 37 °C the cells were treated for different time with the IC50s of 5-FU and DOXO. The oxidative stress was analysed by Hydroethidine (HE) staining after 48 h of treatment. Hydroethidine is used as a vital dye in fluorescence assays that operates as a probe for measurement of O2 −.