Cell Apoptosis Analysis Kit (Hoechst staining)

Adherent cells

1. Remove culture medium and gently wash the cells once with PBS.

2. Add enough Fixative Solution to cover the cells and fix for approximately 10 min at room temperature.

3. Remove the Fixative Solution and wash the cells twice with PBS.

4. Add enough Hoechst 33258 Stain to cover the cells and stain for approximately 5 min at room temperature protected from light.

5. Remove the staining solution and wash twice with PBS.

6. Apply an appropriate amount of Antifade Mounting Medium, place a coverslip while avoiding bubbles, and observe nuclear morphology by fluorescence microscopy.

Suspension cells

1. Collect cells by centrifugation and wash with PBS.

2. Resuspend in Fixative Solution and fix for approximately 10 min. Centrifuge to remove the fixative and wash with PBS.

3. Resuspend cells in Hoechst 33258 Stain and stain for approximately 5 min at room temperature protected from light.

4. Wash with PBS, place cells on a glass slide, add Antifade Mounting Medium, cover, and observe.

Note: Hoechst 33258 bound to dsDNA has excitation/emission maxima of approximately 352/461 nm. Condensed chromatin in apoptotic cells usually shows stronger blue fluorescence.