Induction of Apoptosis by Camptothecin
Materials
1. Prepare a 1.0 mM stock solution of Camptothecin (Sigma-Aldrich, Cat. No. C-9911) in DMSO. 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.
2. Jurkat cells (Human T-cell leukemia; ATCC® TIB-152™).
Note: Mouse thymocytes or other cell types or cell lines can similarly be treated by culturing an agent that induces apoptosis such as Dexamethasone as shown.
Procedure
1. Add camptothecin (4-6 µM final concentration) to 1 × 10^6 /ml proliferating Jurkat cells.
2. Incubate the cells for 4 hr at 37°C.
Active Caspase-3 Staining Protocol
Procedure
1. Determine total amount of experimental samples (tests) and calculate the amount of BD Perm/Wash™ buffer (1×) and antibody you will need so that each test will have 100 µl BD Perm/Wash™ buffer (1×) and 20 µl antibody as follows:
No. Tests No. Cells Perm/Wash™ Vol. (ml) Antibody Vol. (μl)
1 1 × 10^6 0.1 20
5 5 × 10^6 0.5 100
10 10 × 10^6 1.0 200
20 20 × 10^6 2.0 400
2. Dilute the needed amount of BD Perm/Wash™ buffer (10×) 1:10 in distilled water prior to use.
Note: Precipitate may be occasionally observable with the BD Perm/Wash™ buffer (10×) which will not affect performance of the buffer. The precipitate may be removed by filtering the 1× solution through a 0.45 µm filter.
3. Wash cells twice with cold 1× PBS, then resuspend cells in BD Cytofix/Cytoperm™ solution at a concentration of 1 × 10^6 cells/0.5 ml.
4. Incubate cells for 20 min on ice.
5. Pellet cells, aspirate, and discard BD Cytofix/Cytoperm™ solution; wash twice with BD Perm/Wash™ buffer (1×) at a volume of 0.5 ml buffer/1 × 10^6 cells at room temperature.
6. Resuspend cells in the above calculated BD Perm/Wash™ buffer (1×) plus antibody and incubate for 30 min at room temperature.
7. Wash each test in 1.0 ml BD Perm/Wash™ buffer (1×), then resuspend the test in 0.5 ml BD Perm/Wash™ buffer (1×) and analyze by flow cytometry.
Danger: Cytofix/Cytoperm Fixation and Permeabilization Solution (1X) (component 51-6896KC) contains 4.2% formaldehyde.
Hazard statements
Harmful if inhaled.
Causes skin irritation. Causes serious eye damage.
May cause an allergic skin reaction.
Suspected of causing genetic defects.
May cause cancer.
Route of exposure: Inhalation. May cause respiratory irritation.
Precautionary statements
Wear protective clothing / eye protection.
Wear protective gloves.
Do not breathe mist/vapors/spray.
IF IN EYES: Rinse cautiously with water for several minutes.
Remove contact lenses, if present and easy to do. Continue rinsing.
If skin irritation or rash occurs: Get medical advice/attention.
