BD, 560640, BD Pharmingen™ Alexa Fluor® 700 Mouse Anti-Cleaved PARP (Asp 214)

Reactivity: Human (QC Testing)
Isotype: Mouse IgG1, κ
Immunogen: Human cleaved PARP
Application: Intracellular staining (flow cytometry) (Routinely Tested)
Vol. Per Test: 5 µl
RRID: AB_1727571
Storage Buffer: Aqueous buffered solution containing protein stabilizer and ≤0.09% sodium azide.
Regulatory Status: RUO
Preparation And Storage: Preparation And Storage Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated to Alexa Fluor® 700 under optimum conditions, and unreacted Alexa Fluor® 700 was removed.
Recommended Assay Procedures: Recommended Assay Procedures Flow cytometry: 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 . Materials · Prepare a 1.0 mM stock solution of camptothecin (Sigma-Aldrich; Cat. No. C-9911) in DMSO. · Jurkat cell line (ATCC TIB-152), proliferating, at 1 x 10^6 cells/ml. · Either BD Cytofix/Cytoperm™ Fixation/Permeablization Kit (Cat. No. 554714) or Cytofix/Cytoperm™ solution (Cat. No. 554722) plus Perm/Wash™ buffer (Cat. No. 554723). Procedure 1. Add camptothecin (4-6 µM final concentration) per 1 x 10^6 proliferating Jurkat cells.  If desired, a control aliquot of untreated cells should also be prepared. 2. Incubate the cells for 4-6 hours at 37°C. 3. Wash the cells (camptothecin-treated and control aliquots) twice with cold PBS; then resuspend them in BD Cytofix/Cytoperm™ solution at 2 x 10^6 cells/ml. 4. Incubate the cells for 20 minutes on ice. 5. Pellet the cells, and aspirate and discard the Cytofix/Cytoperm™ solution. 6. Wash the cells twice at room temperature with 0.5 ml Perm/Wash™ buffer per 1 x 10^6 cells, and discard the supernatants. 7. Resuspend the cells in Perm/Wash™ buffer at 10 x 10^6 /ml. 8. Aliquot test samples of 1 x 10^6 cells per 100-µl test. 9. Add 5 µl antibody per test, and incubate for 30 minutes at room temperature. 10. Wash each test in 1.0 ml Perm/Wash™ Buffer and discard the supernatant. 11. Resuspend each test in 0.5 ml Perm/Wash™ Buffer and analyze by flow cytometry.
Product Notices: Product Notices 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). An isotype control should be used at the same concentration as the antibody of interest. Alexa Fluor® 700 has an adsorption maximum of ~700nm and a peak fluorescence emission of ~720nm. Before staining cells with this reagent, please confirm that your flow cytometer is capable of exciting the fluorochrome and discriminating the resulting fluorescence. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR. 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. 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. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.