• Staurosporine (from Streptomyces staurosporeus) is a relatively non-selective protein kinase inhibitor that is often used as a general agent for inducing apoptosis. A stock solution of 2 μM staurosporine should be prepared in culture medium before use.
• This antibody stains HeLa (ATCC CCL-2), A549 (ATCC CCL-185) and U-2 OS (ATCC HTB-96) cells, and it works with either 90% cold methanol or BD Perm/Wash™ buffer permeabilization.
1. Seed the cells in appropriate culture medium at ~10,000 cells per well in a BD Falcon™ 96-well Imaging Plate (Cat. No. 353219), and culture overnight.
2. Add the appropriate concentrations of staurosporine, diluted in 100 μl of culture medium, to the wells and culture for 4 hours.
3. Fix the cells by adding 100 µl of a pre-warmed (37°C) 12% formaldehyde solution to each well and incubating for 30 minutes at room temperature (RT). The final volume is 300 μl per well with a 4% formaldehyde final concentration.
4. Remove the fixative from the wells, and wash the cells twice with 1× PBS.
5. Permeabilize the cells using either -20°C 90% cold methanol (eg. BD Phosflow™ Perm Buffer III, Cat. No. 558050) or BD Perm/Wash™ buffer
(Cat. No. 554723):
a. Add 100 µl of -20°C 90% methanol, BD Phosflow™ Perm Buffer III (Cat. No. 558050), to each well and incubate for 5 minutes.
or b. Add 100 µl of 1× BD Perm/Wash™ buffer to each well and incubate for 30 minutes at RT.
6. Remove the permeabilizer, and wash the wells twice with 100 μl of 1× PBS.
7. Dilute the Alexa Fluor® 647 Mouse anti-Cleaved PARP (Asp 214) antibody conjugate:
a. If the cells were permeabilized with cold methanol, dilute the antibody 1:10 in 1× PBS
or b. If the cells were permeabilized with BD Perm/Wash™ buffer, dilute the antibody 1:10 in 1× BD Perm/Wash™ buffer.
8. Stain the cells by adding 50 µl of the diluted antibody conjugate to each well and incubating for 1 hour at RT or overnight at 4°C, protected
9. Remove the diluted antibody conjugate, and wash the wells twice with 100 μl of 1X PBS. Remove the PBS.
10. To counter-stain the nuclei, dilute Hoechst 33342 Solution (Cat. No. 561908) to 2 μg/ml in 1× PBS, and add 200 μl to each well at least 15 minutes before imaging.
11. View and analyze the cells on an appropriate imaging instrument. Recommended filters for the BD Pathway™ instruments are:
Instrument Excitation Emission Dichroic
BD Pathway 855 620/60 700/75 660 LP
BD Pathway 435 628/40 690/40 FF660
For flow cytometry
• Camptothecin, an extract of the Chinese tree Camptotheca acuminata, has been reported to induce apoptosis in a dose-dependent manner in vitro. A stock solution of 1.0 mM camptothecin (eg, Sigma-Aldrich; Cat. No. C-9911) should be prepared in DMSO before use.
1. Add camptothecin at 4-6 µM final concentration per 1 x 10^6 proliferating Jurkat cells (Human T-cell leukemia; ATCC TIB-152). A control aliquot of untreated cells should also be prepared. Incubate the cells for 4 - 18 hours in a 37°C incubator.
2. Wash the cells twice with cold PBS, resuspend them in BD Cytofix/Cytoperm™ solution at 2 x 10^6 cells/ml, and incubate for 20 minutes on ice.
3. Pellet the cells, and aspirate and discard the BD Cytofix/Cytoperm™ solution.
4. Wash the cells twice at room temperature with 0.5 ml BD Perm/Wash™ buffer per 1 x 10^6 cells, and discard the supernatants.
5. Resuspend the cells in BD Perm/Wash™ buffer at 10 x 10^6 cells/ml, and aliquot test samples of 1 x 10^6 cells per 100-µl test.
6. Add 5 µl Alexa Fluor® 647 Mouse Anti-Cleaved PARP (Asp 214) antibody per test, and incubate for 30 minutes at room temperature, protected from light.
7. Wash each test in 1.0 ml BD Perm/Wash™ Buffer and discard the supernatant.
8. Resuspend each test in 0.5 ml BD Perm/Wash™ Buffer and analyze by flow cytometry.