Annexin V, FITC Apoptosis Detection Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Cells were pretreated and incubated with SC-560, NS-398, U46619, and SQ29548 or transfected with TP small interfering RNA (siRNA) for 24 h		- Both FITC-labeled Annexin V and PI are light sensitive. All staining procedures must be performed without direct exposure to intense light.

Upstream tips
- Cells were pretreated and incubated with SC-560, NS-398, U46619, and SQ29548 or transfected with TP small interfering RNA (siRNA) for 24 h
Downstream tips
- Both FITC-labeled Annexin V and PI are light sensitive. All staining procedures must be performed without direct exposure to intense light.

Publication protocol

Apoptosis was assessed with an annexin V-FITC/PI apoptosis detection kit (Dojindo) using BD FACSAria flow cytometry. Briefly, RPMI-8226 and U-266 cells were seeded into 12-well plates and incubated with SC-560, NS-398, U46619, and SQ29548 or transfected with TP small interfering RNA (siRNA) for 24 h. Then the cells were harvested and washed once in PBS and once in 1× binding buffer. After resuspension in 1× binding buffer, we added 5 μl of annexin V to 100 μl of cell suspension, incubated for 15 min at room temperature, washed, and resuspended the cells in 1× binding buffer. After the addition of 5 μl of PI dye, cells were analyzed by BD FACSAria flow cytometry. In addition, caspase 3 activity was assayed using a caspase 3 activity assay kit (Beyotime) according to the manufacturer's instructions.

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Download the product protocol from Dojindo for Annexin V, FITC Apoptosis Detection Kit below.

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