Annexin 5 fluorescein isothiocyanate fitc and propidium iodide apoptosis detection kit

Cellular apoptosis rate was determined using the annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) apoptosis detection kit (BD Biosciences, San Diego, CA) according to the manufacturer's protocol. Briefly, A2780, SKOV3, and A2780/DDP cells were seeded in 6-well plates and exposed to matrine with different concentrations for 24 h. On the following day, cells were harvested, washed twice with cold PBS, resuspended in 500 μL binding buffer and stained with 5 μL FITC-conjugated Annexin V and 10 μL PI for 10 min at 37 °C in the darkness. Fluorescence signals from at least 10,000 cells were analyzed immediately using a FACSCalibur flow cytometry (BD Biosciences, USA). Dot plots and histograms were analyzed by FlowJo software.

On the day of the experiment, U266 cells were harvested and washed two times with RPMI 1640 (Gibco-BRL). The cells were induced apoptosis by seeded in the wells of 24-well culture plates in RPMI-1640 medium (Gibco-BRL) supplemented with 10% (v/v) FBS (Gibco-BRL) and 1% (w/v) PS, in the presence of chaetocin (Sigma-Aldrich) in a dose-dependent manner (25 to 400 nM) for 24 hours, or by high-dose UVB irradiation (120 mJ/cm²) (International light, Newburyport, MA) followed by overnight culture in RPMI-1640 (Gibco-BRL). Cell apoptosis was detected by an Annexin-V fluorescein isothiocyanate (FITC) and propidium iodide (PI) apoptosis detection kit according to the manufacturer's protocol (BD Biosciences, Franklin Lakes, NJ, USA). Next, the samples were acquired on a FACS Calibur cell sorter (Becton Dickinson, Mountain View, CA, USA), and the data were analyzed using WinMDI software (ver. 2.9; Biology Software Net: http://en.bio-soft.net/other/WinMDI.html). The cells were washed extensively before loading onto DCs at a ratio of 2:1 (DCs to dying tumor cells). In addition, the expression of inducible HSP90 in dying tumor cells treated with chaetocin was analyzed by flow cytometry using specific monoclonal antibodies (mAbs) for HSP90 (Stressgen, Ann Arbor, MI, USA).

The percentage of apoptosis was determined using annexin-V fluorescein isothiocyanate (FITC) and propidium iodide (PI) apoptosis detection kit (BD Biosciences) according to the manufacturer’s instructions. Percentage of apoptotic cells (annexin+/PI+) was analyzed by flow cytometry (BD Biosciences).
The distribution of cell cycle was determined by staining permeabilized cells with PI dye after treatment with EP (Sigma) or Dasatinib (Selleckchem) for 24 h. Cells were fixed and permeabilized with 70% alcohol at 4 ℃ and then washed twice with PBS. Cells were incubated with RNase A for 30 min and then stained with PI at room temperature in the dark. For Ki67 expression, cells were permeabilized and fixed, then washed with PBS. Cells were incubated with anti-Ki67-FITC or FITC isotype (BD Biosciences) for 1 h at 4 ℃. Before flow cytometry analysis, 10 μg/ml of PI was added to the cell suspension. Nuclear DNA content was assessed with PI and analyzed by flow cytometry (BD Biosciences). Ki67 negative cells were defined as G0 cells^{9 (link)}.