Annexin 5 fitc and 7 aminoactinomycin d 7aad

Human colorectal cancer cell lines SW620, LoVo, Hct116, Hct8, SW480, HT29 and murine colorectal cancer CT26 were obtained from the American Type Culture Collection. Cells were maintained in a 5% CO₂ humidified atmosphere at 37 °C and cultured in DMEM or RPMI-1640 supplemented with 10% FBS (Dutscher). Cells were routinely tested for mycoplasma contamination using the Mycoalert Mycoplasma Detection Kit (Lonza).
Treatments were carried out for 6, 16, 24, 48 or 72 h with 10 µM of 5-fluorouracil (Sigma Aldrich), oxaliplatin (Accord Healthcare Limited), triacsin C (Cayman Chemical) LPCAT2 inhibitor: TSI-01 (Cayman Chemical), DGAT2 inhibitor: PF-06424439 (Sigma Aldrich), or oleic acid (Sigma Aldrich). Cell viability was determined using the annexin V-FITC and 7-aminoactinomycin D (7AAD) staining from BD Biosciences according to the manufacturer’s instructions. The inhibitory concentrations 50% (IC₅₀) were assessed by crystal violet staining after 48 h of treatment. IC₅₀ values were calculated by a four-parameter non-linear regression with SigmaPlot version 6 software (Systat software, Inc.).

Apoptosis was determined using annexin V-FITC and 7-aminoactinomycin D (7AAD) staining from BD Biosciences (Le Pont de Claix, France) according to the manufacturer’s instructions. Cells were seeded in triplicate into 6-well plates (1.5 × 10⁵ cells per well) 24 h before treatment. The following day, 4T1 cells were untreated (Co) or treated with 6 µg/mL of E.A. and 5 or 10 µM CDDP for 48 h. For E.A./CDDP combination, 4T1 cells were pretreated with 6 µg/mL of E.A. for 24 h, then with 5 µM or 10 µM CDDP for the last 48 h. At the end of treatment, cells were harvested, washed twice with cold PBS, and then incubated for 15 min at room temperature with a mixture of annexin V-FITC/7AAD (5 µL each per sample) in 50 µL of binding buffer. At the end of the incubation, 100 µL of binding buffer was added, and cells were kept on ice until analysis. Annexin V binding and 7AAD incorporation were detected with an LSRII flow cytometer (BD Biosciences, Le Pont de Claix, France) and analyzed using FlowJo software (Tristar, Ashland, OR, USA).

For cell apoptosis analysis, cells were stained with Annexin V-FITC and 7-aminoactinomycinD (7-AAD) (BD Biosciences) for 15 min 48 hours after transfection and subsequently analyzed using a flow cytometer (BD FACSVerse).