Camptothecin, a cytotoxic plant alkaloid with antitumor properties, is a prototypic DNA topoisomerase I inhibitor. Use camptothecin to create positive controls in apoptosis detection experiments.
Camptothecin is a potent cytotoxic pentacyclic alkaloid known for its potent apoptosis inducing properties. Camptothecin and its analogs have been demonstrated to inhibit topoisomerase I (topo I), causing cells in S phase to enter into apoptosis. Camptothecin binds to and stabilizes the topo I – DNA backbone complex (covalent binary complex). This stabilized complex diminishes the rate of topo I release from the broken strand, thus slowing the religation and subsequent DNA synthesis process. Use camptothecin to create positive controls in apoptosis detection experiments.
DNA topoisomerase I
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- Dissolve camptothecin powder in tissue culture grade DMSO to obtain a 2 mg/mL comptothecin stock concentration.
- Prepare 50 – 100 µL aliquots of the DMSO solubilized camptothecin and store them frozen at < -20°C. A frozen vial of camptothecin may be re-thawed 2X before discarding. Vials which have been thawed 1X should be marked to indicate this so that they only go through one more freeze thaw before being discarded.
- Spike cell cultures at a camptothecin concentration range of 2 – 4 µg/mL which yields a cell culture camptothecin concentration of 6 – 12 µM. This camptothecin concentration range works well for inducing Jurkat or HL 60 cell suspensions which usually run in the 1 x 105 – 1 x 106 cell/mL concentration range. At this concentration range successful apoptosis induction has been achieved after a 4 hour (37°C) incubation period. 4. Perform time course studies on your particular cell line to ascertain the optimal camptothecin concentration and exposure time required to achieve good apoptosis induction levels for your experiments. 5. Proceed with your experimental apoptosis induction model system.