BD Pharmingen™ Alexa Fluor® 647 Mouse anti-Cleaved PARP (Asp 214)

For Bioimaging

•        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

       from light.

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.

1. This antibody has been developed and certified for the bioimaging application. However, a routine bioimaging test is not performed on every lot. Researchers are encouraged to titrate the reagent for optimal performance.
2. This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
3. Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
4. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
5. The Alexa Fluor®, Pacific Blue™, and Cascade Blue® dye antibody conjugates in this product are sold under license from Molecular Probes, Inc. for research use only, excluding use in combination with microarrays, or as analyte specific reagents. The Alexa Fluor® dyes (except for Alexa Fluor® 430), Pacific Blue™ dye, and Cascade Blue® dye are covered by pending and issued patents.
6. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
7. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
8. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
9. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.