BestProtocols: Camptothecin Apoptosis Protocol

Cell death cascades associated with apoptosis are complex and dynamic processes that can be assessed by a variety of assay types. The use of a positive experimental apoptosis control provides a level of confidence in the data and is used to ensure the experiment was performed correctly. A positive apoptosis control is not always necessary, but it is good laboratory practice to include. Described is a method of apoptosis induction as a positive control for cell death assays, specifically a method for inducing apoptosis using camptothecin.
 

Materials

• A cell line or primary cells susceptible to apoptosis induction.
• RPMI-1640 medium supplemented with 10% FBS
• 1 mM stock solution of camptothecin prepared in DMSO
• Tissue culture flasks or tissue culture plates
   

Experimental procedure

1. Prepare cells in fresh RPMI-1640 medium with 10% FBS at a concentration of 0.5 x 10⁶ cells/mL in desired tissue culture flasks or tissue culture plates.
2. Add an appropriate amount of 1 mM Camptothecin to the cell suspension to achieve a final concentration of 4-6 µM. The negative control should consist of cells maintained in medium with an equivalent dilution of DMSO only.
3. Incubate cells for the amount of time optimal for your cell type in a humidified, 5% CO2 incubator at 37°C. It is recommended that you first do a time course to get an idea of how sensitive you cells are to undergo apoptosis
4. Harvest cells by centrifugation and proceed with appropriate assay to evaluate the induction of apoptosis.
   • Note: Other pharmacological reagents that have been shown to induce apoptosis include: Actinomycin D, Aphidicolin, Cycloheximide, Dexamethasone, 5-Fluorouracil, Hydroxyurea, and Staurosporine.
      

1. Traganos F, Seiter K, Feldman E, Halicka HD, Darzynkiewicz Z (1996) Induction of apoptosis by camptothecin and topotecan. Ann N Y Acad Sci. 13:803:101-10.
2. Morris EJ, Geller HM (1996) Induction of neuronal apoptosis by camptothecin, an inhibitor of DNA topoisomerase-I: evidence for cell cycle-independent toxicity. J Cell Biol. 1996 Aug;134(3):757-70.