Dead Cell Apoptosis Kit with Annexin V FITC and PI

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Cells were cultured in 35 mm2 disks	- After treating cells with Annexin V and PI working solution they are incubated with Annexin-binding buffer on ice

Upstream tips
- Cells were cultured in 35 mm2 disks
Protocol tips
- After treating cells with Annexin V and PI working solution they are incubated with Annexin-binding buffer on ice

Publication protocol

HUVECs were cultured in 35 mm2 disks. After treatments, cells were collected by trypsinization and centrifugation at 1500 rpm for 5 minutes, followed by washing cell pellet twice with cold PBS and resuspending cell pellet in 1* Annexin-binding buffer. Then, cells were added 5 µL Annexin V and 1 µL 100 µg/ml PI working solution in 100 µL cell suspension, and incubated at room temperature for 15 minutes in the dark. After the incubation, 400 µL 1* Annexin-binding buffer was added. After mixing gently, the fluorescence intensity was detected with a flow cytometry (CyAn ADP, Beckman Coulter, USA) at emission at 530 nm and 575 nm and excitation at 488 nm. The percentage of cells stained by Annexin V/PI which indicates early apoptosis was shown in bar chart.

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