FITC Annexin V Apoptosis Detection Kit I (RUO)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Cells were treated with 1 μM 3-Cl-AHPC and AHP3 for various indicated time.	- Treated with FITC Annexin V and PI and incubated for 15 min at RT (25°C) in the dark

Upstream tips
- Cells were treated with 1 μM 3-Cl-AHPC and AHP3 for various indicated time.
Protocol tips
- Treated with FITC Annexin V and PI and incubated for 15 min at RT (25°C) in the dark

Publication protocol

Pancreatic cancer cell lines were treated with 1 μM 3-Cl-AHPC and AHP3 for various indicated time. Apoptosis in cells was analyzed by flow cytometry using Annexin V-FITC binding together with propidium iodide (PI) staining (Annexin V-FITC apoptosis Detection Kit 1, BD Biosciences, San Diego, CA). Data acquisition was done on a FACS Calibur flow cytometer (BD) and analyzed with CellQuest software (BD Biosciences). 3-Cl-AHPC- and AHP3-mediated inhibition of cell growth were determined by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cells were seeded on 96-well plates at a density of 4 × 104 cells/well in a volume of 200 μL culture medium. 1 μM AHP3 and 3-Cl-AHPC in DMSO (final concentration 0.1%) were added to the cells for various times. 25 μL/well of MTT (5 mg/mL) was added to the medium and incubated for 4 h. After discarding the medium, MTT precipitates were solubilized with 200 μL DMSO and the plates read on a BioTeK Synergy HT (BioTeK Instrument Inc., Vermont) at an absorbance 570 nm. All experiments were performed in quadruplicate to determine means and standard deviations. Cell apoptosis was assessed using acridine orange/ethidium bromide staining as described [10]. The spheres were stained with acridine orange/ethidium bromide staining and immediately visualized and photographed with fluorescence microscope (OLYMPUS CKX41). For DAPI staining, the spheres were incubated with DAPI stain for 30 minutes at 37°C. Then spheres were visualized and photographed with a fluorescence microscope.

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