Annexin V-mCherry/SYTOX Green Apoptosis Detection Kit 

Annexin V-mCherry/SYTOX Green Apoptosis Detection Kit provides a rapid and convenient method to detect cell apoptosis and necrosis. In normal live cells, Phosphatidylserine (PS) is located on the cytoplasmic surface of the cell membrane. Upon initiation of apoptosis, PS is translocated from the inner to the outer leaflet of the membrane. Annexin V is a 35-36 kDa Ca2+-dependent phospholipid-binding protein that has a high affinity for PS. Annexin V labeled with mCherry can identify apoptotic cells by binding to PS exposed on the outer leaflet. SYTOX Green is an excellent green-fluorescent nuclear and chromosome counterstain that only works on dead cells. After staining cells with Annexin V-mCherry and SYTOX Green, live cells show little or no fluorescence, apoptosis cells show red fluorescence, necrosis cells show red and green fluorescence.

-20°C, 1 year

Protect from light

Avoid repetitive freeze-thaw cycles

1. Collect 1-5 × 10⁵ cells

For suspension cells: Centrifuge at 1000 g for 5 minutes and then discard the supernatant. Add 1 mL of pre-cooled PBS to resuspend the cells, centrifuge at 1000 g for 5 minutes and then discard the supernatant.

For adherent cells: Collect the cell culture medium. Wash cells with PBS and add trypsin to dissociate cells. Add the medium and gently suspend the cells to make a single-cell suspension. Centrifuge at 1000 g for 5 minutes and then discard the supernatant. Add 1 mL of pre-cooled PBS to resuspend the cells, centrifuge at 1000 g for 5 minutes and then discard the supernatant.

Note: It is recommended to use trypsin containing no EDTA.

2. Resuspend the cells in 195 μL of Binding Buffer.

3. Add 5 μL of Annexin V-mCherry.

4. Add 1 μL of SYTOX Green.

5. Incubate the cells at room temperature for 10-20 minutes in the dark.

6. Detect the fluorescence by flow cytometer or fluorescence microscope. The maximum excitation wavelength and emission wavelength of mCherry were 587 nm and 610 nm respectively, the maximum excitation wavelength and emission wavelength of SYTOX Green-DNA were 504 nm and 523 nm respectively.