Pe annexin 5 apoptosis detection kit 1

The ESCC cells were seeded in 6-well plates, cultured in RPMI 1640 medium supplemented with 10% FBS for 24 h and then treated with cisplatin for 24 h. The apoptotic cells were double-labeled with annexin V–fluorescein isothiocyanate (FITC) and propidium iodide (PI) using a BD Pharmingen^™ PE Annexin V Apoptosis Detection Kit I (Becton Dickinson FACSCanto II, NJ, USA) according to the manufacturer's instructions.

Duodenal, jejunal and ileal epithelial cells were isolated, to measure the proportion of apoptotic cells by flow cytometry with a PE Annexin V Apoptosis Detection Kit I (Becton, Dickinson and Company, BD Biosciences, San Jose, CA, USA) [27 (link)]. Briefly, the excised mucosal layer of the duodenum, jejunum and ileum were isolated, and then, ground and filtered to form a cell suspension. The cells were carefully washed twice with ice-cold PBS and suspended in the PBS at 1 × 10⁶ cells/mL. After adding 5 μL of PE Annexin V and 5 μL of 7-aminoactinomycin D (7-AAD) to a 100-μL aliquot of the cell suspension, the mixture was incubated at room temperature for 15 min in a dark room. Afterwards, 400 μL of Annexin V Binding Buffer (1×) was added, and the apoptotic cells were examined by flow cytometry (CytoFlex, Beckman Coulter, Inc., Brea, CA, USA) within 1 h.

The proportion of apoptotic cells in isolated jejunal mucosal cells was determined by flow cytometry (CytoFlex, Beckman Coulter, Inc., Brea, CA, USA) using PE Annexin V Apoptosis Detection Kit I (Becton, Dickinson and Company, BD Biosciences, San Jose, CA, USA). First, the jejunum was dissected, the jejunal mucosa was scraped, and then filtered through a grind and a mesh to form a cell suspension. After washing twice with ice-cold PBS, the cell sample was made into a single cell suspension of 1 × 10⁶ cells/mL. One hundred microlitre of the single cell suspension was centrifuged at 1,300 × g for 15 min to remove the supernatant, then the cells were stained with 5 μL of Annexin-V-FITC fluorescent dye at 4°C in the dark. After 10 min, add 5 μL of PI staining for 5 min at 4°C in the dark. Finally, detection of apoptotic cells was completed within 1 h after the addition of 400 μL Annexin V binding buffer (1x).

Cell apoptosis was assessed by flow cytometry with PE Annexin V Apoptosis Detection Kit I (Becton Dickinson Biosciences, Franklin Lakes, NJ) according to the manufacturer's instructions. Briefly, cancer cells were seeded in 6‐well plates at a density of 1 × 10⁵ cells per well. After being starved overnight, cells were treated with fresh medium containing various concentrations of TAM for 24 hours. Then cells were trypsinized, washed with phosphate‐buffered saline (PBS), and stained with PE Annexin V. The percentage of apoptotic cells was quantified by flow cytometry using a FACSCalibur instrument (BD Biosciences). The total apoptosis rate was calculated by summing the rate of early apoptotic cells (7‐AAD−/PE Annexin V+) and late apoptotic cells (7‐AAD+/PE Annexin V+).

Platelet apoptosis was calculated from phosphatidylserine (PS) exposure on platelet surface using PE Annexin V Apoptosis Detection Kit I from BD (Becton Dickinson, San Jose, CA). Platelets were washed with PBS-human albumin solution and resuspended in annexin binding buffer. Platelets (1 × 10⁶) were incubated with saturating concentrations of the Annexin-V and 7AAD antibodies and incubated for 15 min in dark conditions. After incubation, platelets were washed with annexin binding buffer and analyzed using a FACSCalibur (Becton Dickinson, San Jose, CA) and CellQuestPro software. Data analysis was performed using FlowJo software (Becton Dickinson, San Jose, CA).