Cell Meter™ Annexin V Binding Apoptosis Assay Kit *Orange Fluorescence Optimized for Flow Cytometry*

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

2. Add Annexin V-iFluor® 555 assay solution.

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

4. Analyze cells using a flow cytometer with a 575/26 nm filter (PE channel) or fluorescence microscope with a Cy3 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® 555 (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 575/26 nm filter (PE channel) or a fluorescence microscope with a Cy3 filter set.

1. Quantify Annexin V- iFluor® 555 binding using a flow cytometer with a 575/26 nm filter (PE 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® 555, 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 incubation with Annexin V-iFluor® 555 and visualized under a microscope.

3. Analyze the apoptotic cells with Annexin V-iFluor® 555 under a fluorescence microscope using a Cy3 filter set. Measure the cell viability by using the Cy5 channel when Nuclear Red™ DCS1 (AAT catalog #17552) is added to the cells. The orange staining on the plasma membrane indicates the Annexin V-iFluor® 555 binding to PS on the cell surface.