Killing Cells with Camptothecin for Positive Control

2015-01-13

azim58 - Killing Cells with Camptothecin for Positive Control

Original Protocol: FITC Active Caspase 3 Apoptosis Kit

Induction of Apoptosis by Camptothecin

* Prepare a 1.0 mM stock solution of camptothecin (Sigma-Aldrich Cat.
  No. C-9911) in DMSO (maybe 1% DMSO is better since this would harm the
  cells less and most likely still dissolve the camptothecin).
  Camptothecin, an extract of the Chinese tree Camptotheca acuminata, is
  a potent inhibitor of topoisomerase I, a molecule required for DNA
  synthesis. Camptothecin has been reported to induce apoptosis in a dose
  dependent manner in vitro.
  o Camptothecin molecular weight: 348.4 g/mol
  o 1 mmol/(1000 mL)* 1 mol/ (1000 mmol) * 348.4 g/(1 mol) * 1 mL =
    3.484*10^(-4)g or 0.348 mg

* Add camptothecin to 1X10e6/mL proliferating Cells. We will probably
  use the max concentration of cells that we can fit in 3 mL.
  Concentration of cells measured using trypan blue.
  o Groups
    - No camptothecin control
    - 0.1 uM
    - 2 uM
    - 6 uM
    - Jurkat (6 uM) (maybe. . . these cells weren't growing well.
      @ Volume 1 mM stock needed
      @ 1mM*y/(1000*uL)=0.0001 mM -> y=0.0001 mM * 1000 uL / (1
        mM)
        # 0.1 uM -> 0.1 uL (of 1 mM into 1 mL)
          2 uM -> 2 uL
          6 uM -> 6 uL

* Incubate the cells for at 37 C
  o The recommended time is 4 hr. However, the incubation time will
    depend on the cell type.
    - We were able to detect killing of Jurkat cells with a 4 hr
      incubation, but no killing of NIH 3T3 fibroblast cells was detected
      with this time. The company recommended using a 24 hour incubation.

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