Pe labeled annexin 5 and 7 aad

Melanoma cells were cultured for 36 or 72 h in the presence of different concentrations of baicalein and baicalin, and apoptosis was analyzed after staining with PE-labeled Annexin V and 7-AAD (BD Biosciences, San Diego, CA, United States) (Liu et al., 2015 (link); Dou et al., 2018 (link); Wang et al., 2018 ). Stained cells were analyzed on a FACSCalibur (BD Bioscience) and the data were analyzed with the FlowJo software (Tree Star, Ashland, OR, United States).

Colon cancer cells were cultured for 72 or 120 hours in the presence of different concentrations of baicalein and baicalin, and apoptosis was analyzed after staining with PE-labeled Annexin V and 7-AAD (BD Biosciences, San Diego, CA, USA), as we previously described [31 (link), 37 (link)]. For cell cycle analysis, treated tumor cells were fixed with 70% ethanol overnight, washed with PBS and incubated with propidium iodide (10 μg/ml) and RNase A (100 μg/ml). Untransfected cells served as a control. Stained cells were analyzed on a FACSCalibur (BD Bioscience) and the data were analyzed with FlowJo software (Tree Star, Ashland, OR, USA). For determination of cell apoptosis in tumor tissues, the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP digoxigenin nick-end labelling (TUNEL) method (Vazyme, China) was utilized. Briefly, paraffin-embedded sections were deparaffinized with xylene and dehydrated, and then added proteinase K for 20 minutes. Slides were further stained with the FITC-labelled TdT and followed with the propidium Iodide (PI) (1 μg/ml) (cell nucleus labeling). The stained cells were examined and counted with a microscope (400 ×).

THP-1 cells or Molm-13 cells in their logarithmic growth phases were added to a 6-well plate with 2 × 10⁶/well and incubated with different concentrations of U73122 for 24 h before cells were collected.
(1) The cells were fixed using 70% pre-cold ethanol overnight and then stained with 400 µl PI (50 µg/ml) and 100 µl RNase A (100 µg/ml) at room temperature for 15 min in the dark. Flow cytometry analysis was performed using Flow Cytometer (BD Biosciences) to determine the percentage of cells at every phase of the cell cycle.
(2) The cells were collected, washed twice using the binding buffer, and incubated with PE-labeled Annexin-V and 7-AAD (BD Biosciences) at room temperature in the dark for 25 min. Cell apoptosis was determined by flow cytometry.