Cell Meter™ Phosphatidylserine Apoptosis Assay Kit *Blue Fluorescence Excited at 405 nm*

Protocol summary

1. Prepare cells with test compounds (200 µL/sample)
2. Add Apopxin™ Violet 450 assay solution
3. Incubate at room temperature for 30 - 60 mintues
4. Analyze cells using flow cytometer with 450/40 nm filter (Pacific Blue channel) or fluorescence microscope with Violet filter

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

Prepare and incubate cells with Apopxin™ Violet 450:

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 Apopxin™ Violet 450 (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 450/40 nm filter (Pacific Blue channel) or a fluorescence microscope with Violet filter.

Analyze by using a flow cytometer:

1. Quantify Apopxin™ Violet 450 binding using a flow cytometer with 450/40 nm filter (Pacific Blue channel). Measure the cell viability using 610/20 nm filter (PE-Texas Red channel)when propidium iodide is added into the cells. Note: Apopxin™ 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 Apopxin™ Violet 450, 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 Apopxin™ Violet 450 and visualized under a microscope.
   
   
3. Analyze the apoptotic cells with Apopxin™ Violet 450 under a fluorescence microscope with Violet filter. Measure the cell viability using TRITC filter when propidium iodide is added into the cells. The blue staining on the plasma membrane indicates the Apopxin™ Violet 450 binding to PS on cell surface.