Annexin 5 fitc staining kit

FRH-0201 cells were seeded at 2×10⁵ cells in a 6 cm cell culture dish and cultured overnight, after which the cells were exposed to one of three different treatments: varying concentrations of lapatinib(0, 1, 5, 20 μM) at 48 hours, constant concentration exposure (20 μM) for different time durations (0, 12, 24, 48 hours) and a combination of a varying concentrations of lapatinib(0, 1, 5, 10 μM) and a constant concentration of gemcitabine(0.5 μM) over a duration of 48 hours. The cell cycle distribution was detected by staining DNA with propidium iodide (C1052 Beyotime), while apoptosis and necrosis were detected using an Annexin V-FITC staining kit (C1062 Beyotime). The cell cycle distribution and apoptosis were determined through flow cytometry (Beckman CytoFLEX).

The Annexin V-FITC staining kit from Beyotime Biotechnology (Shanghai, China) was used to determine and quantify the apoptotic cells using flow cytometry according to the manufacturer’s instruction. In brief, the collected cells were suspended in the supplied binding buffer, and then stained with FITC-conjugated annexin V and PI at room temperature (RT) for 20 min in the dark. The fluorescent intensities of the cells were detected using flow cytometry, and the Annexin V⁺/PI⁻ and Annexin V⁺/PI⁺ cell populations were representative of apoptotic cells.

The AFM1 powder used in this experiment was provided by Pribolab (Qingdao, China). Dissolve AFM1 in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA) to prepare 200 μg/mL stock solution, and then dilute it to the required concentration with culture medium. Lf powder was obtained from Sigma-Aldrich (St. Louis, MO, USA). Dissolve Lf power in culture medium to make 10 mg/mL stock solution, and then dilute it to the required concentration. Nonessential amino acids, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and Opti-MEM Medium were purchased from Gibco (Carlsbad, CA, USA). Trypsin (2.5%), antibiotics (100 units/mL penicillin, 100 μg/mL streptomycin), Nonessential amino acids (NEAA), Enhanced Cell Counting Kit-8 (CCK-8), and Annexin-V-FITC Staining Kit were provided by Beyotime Biotechnology (Shanghai, China). Trizol Reagent Kit and Lipofectamine 2000 were obtained from Invitrogen (Carlsbad, CA, USA). The Prime Script RT Reagent Kit (Perfect Real Time) and the TB Green Premix Ex Taq II (Tli RNaseH Plus) were obtained from Takara (Shiga Prefecture, Japan). The β-actin and caspase9 antibodies were provided by Bioss (Beijing, China); and the Atg5, SQSTM1/p62, LC3B, caspase3 antibodies, and Atg5-specific siRNAs were provided by Cell Signaling Technology (Danvers, MA, USA).