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.