Cell Meter™ Annexin V Binding Apoptosis Assay Kit *Orange Fluorescence Excited at 405 nm*

Protocol summary

1. Prepare cells with test compounds (200 µL/sample)
2. Add Annexin V-mFluor Violet™ 550 assay solution
3. Incubate at room temperature for 30 - 60 mintues
4. Analyze cells using flow cytometer with 525/50 nm filter (Pacific Orange channel) or fluorescence microscope with Violet filter set

Important notes
Thaw 100X Propidium Iodide (Component C) at room temperature before starting the experiment.

Prepare and incubate cells with Annexin V-mFluor Violet™ 550:

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-mFluor Violet™ 550 (Component A) into the cells.
   
   
5. Optional: Add 2 µL of 100X Propidium Iodide (Component C) into the cells for necrosis cells.
   
   
6. Incubate at room temperature for 30 to 60 minutes, protected from light.
   
   
7. Add 300 µL of Assay Buffer (Component B) to increase volume before analyzing the cells with a flow cytometer or fluorescence microscope.
   
   
8. Monitor the fluorescence intensity using a flow cytometer with 525/50 nm filter (Pacific Orange channel) or a fluorescence microscope with Violet filter set.

Analyze by using a flow cytometer:

1. Quantify Annexin V-mFluor Violet™ 550 binding using a flow cytometer with 525/50 nm filter (Pacific Orange channel). Measure the cell viability using 610/20 nm filter (PE-Texas Red channel) when propidium iodide is added into the cells. 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.

Analyze by using a fluorescence microscope:

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-mFluor Violet™ 550, rinse 1 - 2 times with assay buffer, and add assay buffer back to the cover-slip. Invert 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-mFluor Violet™ 550 and visualized under a microscope.
   
   
3. Analyze the apoptotic cells with Annexin V-mFluor Violet™ 550 under a fluorescence microscope using Violet filter set. Measure the cell viability using TRITC channel when propidium iodide is added into the cells. The orange staining on the plasma membrane indicates the Annexin V-mFluor Violet™ 550 binding to PS on cell surface.