Annexin V-FITC Early Apoptosis Detection Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- 5 × 10^4 cells/well were seeded. - 24h post seeding, cells were treated with MINS for 24 h, 36 h or 48 h.	- First cells were stained with propidium iodide (50 μg/mL) in the dark for 60 min at 4˚C. - Next stained with Annexin V-FITC in the dark for 15 min at room temperature. - Lastly, cells were stained with Hoechst 33342 (10 µg/mL) in the dark for 15 min at room temperature

Upstream tips
- 5 × 10^4 cells/well were seeded. - 24h post seeding, cells were treated with MINS for 24 h, 36 h or 48 h.
Protocol tips
- First cells were stained with propidium iodide (50 μg/mL) in the dark for 60 min at 4˚C. - Next stained with Annexin V-FITC in the dark for 15 min at room temperature. - Lastly, cells were stained with Hoechst 33342 (10 µg/mL) in the dark for 15 min at room temperature

Publication protocol

Cell apoptosis was evaluated in HT-29 cells, HCT-116 cells, Caco-2 (p53 null) cells and Caco-2-p53 cells by
high content screening (HCS) (Thermo Scientific Cellomics ArrayScan Vti, Cellomics, Inc., Pittsburgh, PA) analysis. Firstly, cells were seeded into 96-well plates at 5 × 104 cells/well. After 24 h, HT-29 cells were treated with MINS for 24 h, 36 h or 48 h. Then the cells were centrifugated with horizontal centrifuge for 5 min, stained with propidium iodide (50 μg/mL) in the dark for 60 min at 4˚C and washed 3 times with phosphate-buffered saline (PBS). Secondly, cells were stained with Annexin V-FITC in the dark for 15 min at room temperature, then washed 2 times with Annexin V-FITC binding solution. Briefly, Annexin V-FITC stock solution (1 μL) was added to Annexin V-FITC binding solution (20 μL). Thirdly, cells were stained with Hoechst 33342 (10 µg/mL) in the dark for 15 min at room temperature, and washed 3 times with PBS. Finally, 50 μL PBS was added in every well. The analysis was done using Thermo Scientific Cellomics ArrayScan Vti (MA, USA). Because Caco-2-p53 cells in this study had been transfected green flourescence, so we used AnnexinV-PE with red fluorescene and PI, respectively. The experimental procedures referred to the above three staining methods. Annexin V-positive cells with nuclear shrinkage, blebbing, and apoptotic bodies were counted as apoptotic. The percentage of apoptotic cells was calculated after observing a total of 300 cells. For the p53 inhibitor analysis, the cells
were pretreated with PFT-α (10 µM) for 2 h respectively, and then exposed with MINS.

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