Annexin 5 fitc and pi

To determine the effects of Gyp-L on the cell cycle distribution, ECA-109 cells were treated with Gyp-L for 24 h, fixed with 70% (v/v) ethanol, and stained with 50 μg/ml propidium iodide (Sigma, P4170) containing 0.1 mg/ml RNase and 5% Triton X-100 for 30 min at 37°C. The percentage of cells in different phases of the cycle was analyzed using a flow cytometer (BD FACSCalibur, USA) and inbuilt software. For apoptosis analysis, the cells were incubated with various concentrations of Gyp-L for 24 h, harvested, washed, and resuspended in 500 μl of 1× binding buffer containing Annexin V-FITC and PI (BD, 556547) for 10 min at room temperature. Mitochondrial membrane potential and mass were evaluated using the probe JC-1 (Beyotime, C2005), or Mito-Tracker Green (Beyotime, C1048), respectively. Briefly, esophageal cancer cells in each group were harvested, washed twice with cold PBS, centrifuged, and then stained with JC-1 (5 μg/ml) or Mito-Tracker Green (200 nM) for 30 min at 37°C and analyzed using flow cytometry.

Cells were harvested after 48-h transfection and stained with Annexin V/FITC and PI (BD, USA) following the instruction. The results were analyzed using FACSDiva 7.0 software. Annexin V-FITC⁺ and PI⁻ populations indicated apoptosis. Experiments were conducted in triplicates. Annexin V⁻ and PI⁻ populations were healthy cells that were considered negatively stained. Annexin V⁺ and PI⁻ cells indicated cells in early apoptosis. Moreover, Annexin V⁺ and PI⁺ staining indicated cells in necrosis (postapoptotic necrosis or late apoptosis). Three replications were needed for each samples and three independent experiments for each reaction.

Flow cytometry was performed for apoptosis and cell cycle analysis. Cells were trypsinized and harvested at 72 hours post‐irradiation (10 Gy). The collected cells were stained with Annexin V‐FITC and PI following the manufacturer's protocol (BD Biosciences, Franklin Lakes, NJ, USA). For cell cycle distribution analysis, cells were fixed with 70% cold ethanol for 24 h and digested with RNase for 30 min at 37°C at 48 hours after irradiation of 6 Gy. Then cells were stained with propidium iodide (PI) for 30 min at 4°C according to the manufacturer's protocol (Promoter, Wuhan, China). The samples were subjected to flow cytometry using LSRFortessa (BD Biosciences, San Jose, CA), and data were analyzed using FlowJo version 10 (TreeStar, San Diego, CA, USA).

DU-145 and PC-3 prostate cancer cell lines were treated with lycopene extracts, at concentrations of 500 and 5000 μg/mL, in 6-well plates. After 96 h of incubation, the non-adherent cells were collected, and adherent cells were quickly washed with PBS and detached with trypsin/EDTA(ethylenediamine tetraacetic acid) 0.125% (Sigma Chemical Co., Saint Louis, MO, USA) at room temperature. Cells were centrifuged to remove the medium, washed with PBS and stained with Annexin V-FITC and PI in binding buffer (BD Pharmingen) according to the manufacturer’s instructions. Stained cells were analyzed using a FACSCalibur (BD Bioscience, Franklin Lakes, NJ, USA) and analyzed using WinMDI 2.9 software. Data were reported as the percentage of apoptosis, obtained by determining the numbers of apoptotic cells versus the total numbers of cells.

FCM analysis was performed to detect cell apoptosis using the Annexin V-FITC/PI apoptosis detection kit (Beyotime Institute of Biotechnology, China). Transfected cells were collected following trypsinization and resuspended in Annexin V-FITC Binding Solution. The cell suspension (100 µL) was cultured with 5 µL annexin V-FITC and PI (BD Biosciences, USA) according to the manufacturer’s protocol. Stained cells were counted using a FACSCalibur flow cytometer (BD Biosciences, USA) and Kaluza Analysis (version 2.1.1.20653; Beckman Coulter, Inc., USA).

Bone marrow cells were cultured in 10% FBS RPMI 1640 medium (Life Technologies) for 48 hours, washed and stained for Ly-6G-APC and Annexin V FITC and PI (BD Biosciences) and analyzed by flow cytometry.

Cells were digested and collected by trypsin without EDTA after drug treatment. Each sample was added 5 μL Annexin V-FITC and PI (BD, 556,547) for 15 min in the dark. The apoptotic ratio was analyzed by flow cytometry (BD, LSRFortessa). Apoptosis was calculated as the ratio of early (Q4) and late (Q2) apoptotic cells.