eBioscience™ Annexin V-FITC Apoptosis Detection Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Cells were treated with LfcinB-P13 (range, 0–60 µg/ml) for 24 h.	- Treated with AV, PI and incubate 10-15 minutes at room temperature.	- Due to the calcium dependence of the Annexin V:PS interaction, it is critical to avoid buffers containing EDTA or other calcium chelators during Annexin V experiments. - Annexin V can only be used as a marker of apoptosis in cells where the plasma membrane is intact because destroying the integrity of the plasma membrane will allow non-specific binding of Annexin V to PS inside the cell.

Upstream tips
- Cells were treated with LfcinB-P13 (range, 0–60 µg/ml) for 24 h.
Protocol tips
- Treated with AV, PI and incubate 10-15 minutes at room temperature.
Downstream tips
- Due to the calcium dependence of the Annexin V:PS interaction, it is critical to avoid buffers containing EDTA or other calcium chelators during Annexin V experiments. - Annexin V can only be used as a marker of apoptosis in cells where the plasma membrane is intact because destroying the integrity of the plasma membrane will allow non-specific binding of Annexin V to PS inside the cell.

Publication protocol

The apoptotic effect of LfcinB-P13 on SMMC7721 cells was detected using an Annexin V-FITC (AV-FITC) and propidium iodide (PI) Apoptosis Detection kit (eBioscience, Inc., San Diego, CA, USA). SMMC7721 cells were incubated at 37°C with multiple concentrations of LfcinB-P13 (range, 0–60 µg/ml) for 24 h. Treated cells were harvested via trypsin digestion, washed twice with PBS and subsequently used in the AV/PI assay, which was performed according the manufacturer's protocol. Assayed cells were analyzed using a flow cytometer (BD Biosciences, San Jose, CA, USA). AV-FITC signals were measured at 635 nm for excitation and 660 nm for emission, and PI signals were measured at 535 nm for excitation and 615 nm for emission. Cell population fractions in four quadrants were analyzed by performing quadrant statistics using FlowJo (version 7.6; Tree Star, Inc., Ashland, OR, USA) software. Early and late apoptotic cells were observed in the lower and upper right quadrants, respectively. The percentage of apoptotic cells was calculated using the following formula: Apoptotic rate (%) = (number of apoptotic cells / total number of cells observed) × 100.

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Manufacturer protocol

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