Annexin V, PacBlue conjugate

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

2. Add Annexin V conjugate assay solution.

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

4. Analyze with a flow cytometer or a fluorescence microscope.

The fluorescent annexin V conjugates are stored in a PBS buffer solution containing 0.1% bovine serum albumin (BSA) with a pH of 7.4. To ensure their stability, it is recommended that the solutions be stored at a temperature of -20°C and protected from light. Avoid exposing the fluorescent conjugates to repeated freeze-thaw cycles as this can have a negative effect on their integrity. These solutions can be stored for at least 6 months under the recommended conditions.

1. Prepare an Annexin V-binding assay buffer: 10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2, pH 7.4.

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

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

4. Resuspend cells in 200 μL of the Annexin V-binding assay buffer from Step 1.

5. Add 2 μL of the Annexin V conjugate to the cells.

   Optional: Add a dead cell stain such as Propidium Iodide (Cat No. 17585) into the cells for necrosis cells.

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

7. Add 300 μL of the Annexin V-binding assay buffer (from Step 1) to increase volume before analyzing the cells with
   a flow cytometer or fluorescence microscope.

8. Monitor the fluorescence intensity by using a flow cytometer or a fluorescence microscope.

1. Quantify Annexin V conjugates binding by using a flow cytometer with appropriate filters.

   Note: It is not common to perform Annexin V binding flow cytometric analysis on adherent cells due to the possibility of membrane damage during cell detachment or harvesting. However, previous studies by Casiola-Rosen et al. and van Engelend et al. (refer to Refs 1 and 2) have demonstrated methods for using Annexin V in flow cytometry on adherent cell types.

1. Pipette the cell suspension from Step 6, rinse 1-2 times with Annexin V-binding assay buffer (Step 1), and then resuspend the cells with the Annexin V-binding assay buffer (Step 1).

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. 

3. After incubation with Annexin V conjugate (Step 6), rinse 1-2 times with Annexin V-binding assay buffer (Step 1), and add
   Annexin V-binding assay buffer (Step 1) back to the cover slip.

4. 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 conjugate and visualized under a microscope with the appropriate filter set.

1. Pascal Clerc, Pauline Jeanjean, Nicolas Halalli, Michel Gougeon, Bernard Pipy, Julian Carrey, Daniel Fourmy, Véronique Gigoux. Journal of Controlled Release (2017).

2. Hanshaw RG, Lakshmi C, Lambert TN, Johnson JR, Smith BD. Chembiochem, 6, 2214. (2005).