Fitc conjugated annexin 5

Exponentially growing cells were seeded in 25 cm² flasks and, 24 h later, they were exposed to different concentrations of ACY1215, AZD6244, paclitaxel or to the double (ACY1215, AZD6244) and triple (ACY1215, AZD6244, paclitaxel) combination of drugs for 48 h. For the double combination a 24 h pre-incubation with ACY1215 followed by a 24 co-incubation of ACY1215 and AZD6244 was also tested. At the end of treatment, floating and adherent cells were harvested for detection of apoptotic cells by Annexin V-binding assay (Immunostep, Salamanca, Spain). Cells were washed with cold PBS and re-suspended in binding buffer (10 mM HEPES-NaOH, pH 7.4, 2.5 mM CaCl₂, and 140 mM NaCl, Immunostep). A fraction of 10⁵ cells was incubated in binding buffer at room temperature in the dark for 15 min with 5 μL of FITC-conjugated Annexin V and 10 μL of 2.5 μg/mL propidium iodide (Immunostep). Annexin V binding was detected by flow cytometry. At least 10⁴ events/sample were acquired and analyzed using specific software (CellQuestPro, Becton Dickinson).

Apoptosis was evaluated by Annexin V-binding assay (Immunostep, Salamanca, Spain) in cells treated for 48 h with PLX4032, alone or in combination with orlistat or U18666 A, according to a simultaneous schedule. At the end of treatment, floating and adherent cells were harvested, washed with cold PBS and re-suspended in binding buffer (10 mM HEPES-NaOH, pH 7.4, 2.5 mM CaCl2, and 140 mM NaCl, Immunostep). A fraction of 10⁵ cells was incubated in binding buffer at room temperature in the dark for 15 min with 5 μL of FITC-conjugated Annexin V and 10 μL of 2.5 μg/mL propidium iodide (Immunostep, Salamanca, Spain). Annexin V binding was detected by flow cytometry. At least 10⁴ events/samples were acquired and analyzed using a specific software (CellQuestPro, BD Biosciences, Franklin Lakes, NJ, USA). The activation of caspase 8 and 3/7 was determined using luminescent Caspase Glo 8 and 3/7 assays (Promega, Fitchburg, WI, USA). Cells were seeded (8 × 10³ cells/well) in 96-well plates and treated with drugs for 72 h. Caspase activation was detected according to manufacturer’s instructions.

Apoptosis was evaluated by Annexin V-binding assay (Annexin V-FITC kit, Immunostep, Salamanca, Spain) 24 h after seeding in untransfected cells, and cells transfected with siRNA or negative control oligonucleotides, treated for 72 h with cisplatin. After washing with cold PBS and resuspension in binding buffer (10 mM Hepes-NaOH, pH 7.4, 2.5 mM CaCl₂, and 140 mM NaCl, Immunostep), 10⁵ cells were incubated in binding buffer at room temperature in the dark for 15 min with 5 µl of FITC-conjugated Annexin V and 10 μl propidium iodide (Immunostep). Flow cytometry (BD Accuri, Becton Dickinson) was used for Annexin V binding detection, by acquiring 10⁴ events/sample. Data were analyzed using the Cell- QuestPro software (Becton Dickinson). The activation of caspases was determined using luminescent Caspase Glo 3/7 and Caspase Glo 8 assays (Promega, Fitchburg, WI, United States). Cells were seeded (8 × 10³ cells/well) in 96-well plates and treated with cisplatin for 48 h. Caspase activation was detected according to manufacturer’s instructions.