Cell Meter™ Annexin V Apoptosis Assay Kit

1. Prepare cells with test compounds (200 µL/sample).

2. Add Annexin V-iFluor® 647 assay solution.

3. Incubate at room temperature for 30 - 60 minutes.

4. Analyze cells using a flow cytometer with a 660/20 nm filter (APC channel) or fluorescence microscope with a Cy5 filter set.

For guidelines on cell sample preparation, please visit:

https://www.aatbio.com/resources/guides/cell-sample-preparation.html

1. Treat cells with test compounds for a desired period of time (4 - 6 hours for Jurkat cells treated with staurosporine) to induce apoptosis.

2. Centrifuge the cells to get 1 - 5×10⁵ cells/tube.

3. Resuspend cells in 200 µL of Assay Buffer (Component B).

4. Add 2 µL of Annexin V-iFluor® 647 (Component A) into the cells.

5. Incubate at room temperature for 30 to 60 minutes, protected from light.

6. Add 300 µL of Assay Buffer (Component B) to increase volume before analyzing the cells with a flow cytometer or fluorescence microscope.

7. Monitor the fluorescence intensity using a flow cytometer with a 660/20 nm filter (APC channel) or a fluorescence microscope with a Cy5 filter set.

1. Quantify Annexin V-iFluor® 647 binding using a flow cytometer with a 660/20 nm filter (APC channel).

   Note: Annexin V binding flow cytometric analysis on adherent cells is not routinely tested since specific membrane damage may occur during cell detachment or harvesting. However, methods for utilizing Annexin V for flow cytometry on adherent cell types have been previously reported by Casiola-Rosen et al. and van Engelend et al.

1. Pipette the cell suspension after incubation, rinse 1 - 2 times with Assay Buffer, and then resuspend the cells with assay buffer.

2. Add the cells on a glass slide that is covered with a glass cover-slip.

   Note: For adherent cells, it is recommended to grow the cells directly on a cover-slip. After incubation with Annexin V-iFluor® 647, rinse 1 - 2 times with assay buffer, and add assay buffer back to the cover-slip. Invert the cover-slip on a glass slide and visualize the cells. The cells can also be fixed in 2% formaldehyde after the incubation with Annexin V-iFluor® 647 and visualized under a microscope.

3. Analyze the apoptotic cells with Annexin V-iFluor® 647 under a fluorescence microscope using the Cy5 filter set. The orange staining on the plasma membrane indicates that Annexin V-iFluor® 647 has bound to PS residues on the cell surface.