The levels of cell surface phosphatidylserine were determined to monitor the type of cell death and the kinetics of apoptosis [17 (link)]. The CRC cell lines SW1116 and SW837 were seeded (2.5 × 105 cells/well) into 24-well plates and incubated in a non-CO2 incubator at 37˚C for 18 h. Colon cancer (SW1116) cells were simultaneously treated with Sora (5 µM) and Cur or Que (200 and 400 µM) for 72 h, and rectum cancer (SW837) cells were treated with Sora (5 µM) and Cur or Kmf (200 and 400 µM). The cells were subsequently washed twice with HBSS and harvested using trypsin. Finally, the cells were double-stained using the Annexin V-FITC-Flous staining kit, according to the manufacturer’s instructions (Roche Diagnostic GmbH). Annexin V-Flous labelling solution containing Annexin V-FITC and PI (100 µl) was added to both the treated and control cell groups and incubated at 15–20˚C for 15 min. The cells (1 × 106 cells/ml) were then resuspended in binding buffer, and fluorescence was monitored using flow cytometry (FC500; Beckman Coulter, Inc.).
Annexin 5 fitc flous staining kit
Manufactured by Roche
The Annexin V-FITC-Flous staining kit is a laboratory tool used to detect and quantify apoptosis, a programmed cell death process. The kit contains Annexin V, a protein that binds to phosphatidylserine, a molecule that is exposed on the surface of cells undergoing apoptosis. The Annexin V is labeled with a fluorescent dye, FITC-Flous, which allows the visualization and measurement of apoptotic cells using flow cytometry or fluorescence microscopy.
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2 protocols using annexin 5 fitc flous staining kit
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Apoptosis Kinetics in Colon and Rectum Cancer
2
Annexin V-FITC Apoptosis Assay
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The apoptotic cells were estimated by determing the levels of phosphatidylserine on cell surface. HepG2 and Hep3b cells were seeded into 24-well plates at 2.5×105 cells/well and incubated in a CO2 incubator at 37°C for 18 h. The cells were then simultaneously treated with Cur and Kmf (200 or 400 μM) and Sora (5 μM) for 72 h, washed twice with HBSS, harvested by trypsinization and washed. Finally, the cells were double-stained using the Annexin V-FITC-FLOUS staining kit according to manufacturer's instructions (Roche Diagnostics GmbH, Mannheim, Germany). In brief, Annexin V-FLOUS labeling solution containing Annexin V-FITC and PI (100 μl) was added to treated and control cell groups, followed by incubation at 15-20°C for 15 min. The cells (1×106 cells/ml) were then re-suspended in binding buffer and fluorescence was monitored by flow cytometry (FC500; Beckman Coulter).
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