Cell Meter™ Annexin V Apoptosis Assay Kit

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

2. Add Annexin V-iFluor® 488 stock solution.

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

4. Analyze cells using a flow cytometer with a 530/30 nm filter (FITC channel).

Before starting the experiment, thaw the vial of 100X Propidium Iodide (Component C) at room temperature.

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 camptothecin) to induce apoptosis.

   Note: Annexin V 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.

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® 488 (Component A) into the cells.

5. Optional: Add 2 µL of 100X Propidium Iodide (Component C) for necrosis cells.

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

7. Optional: Add 200 to 300 µL of Assay Buffer (Component B) to increase volume before analyzing the cells with a flow cytometer.

8. Monitor the fluorescence intensity of Annexin V-iFluor® 488 using a flow cytometer with a 530/30 nm filter (FITC channel). Measure the cell viability using the 610/20 nm filter (PE-Texas Red channel) after adding propidium iodide to the cells.