BD, 558576, BD Pharmingen™ FITC Mouse anti-Cleaved PARP (Asp214)

Alternative Name: Asp214
Reactivity: Human (QC Testing), Mouse (Tested in Development)
Isotype: Mouse IgG1, κ
Immunogen: Human cleaved PARP
Application: Intracellular staining (flow cytometry) (Routinely Tested)
Vol. Per Test: 20 µl
RRID: AB_647179
Storage Buffer: Aqueous buffered solution containing BSA 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 with FITC under optimum conditions, and unreacted FITC was removed.
Recommended Assay Procedures: Recommended Assay Procedures 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 shown to induce apoptosis in a dose dependent manner in vitro . Camptothecin is used at BD Biosciences Pharmingen as a general method for inducing apoptosis. Materials • 1.0 mM stock solution of Camptothecin (Sigma; 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 BD Cytofix/Cytoperm™ solution (Cat. No. 554722) plus BD Perm/Wash™ buffer (Cat. No. 554723). Procedure 1. Add Camptothecin (4-6 μ M 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 BD Cytofix/Cytoperm™ solution. 6. Wash the cells twice at room temperature with 0.5 ml BD Perm/Wash™ buffer per 1 x 10^6 cells, centrifuge and discard the supernatants. 7. Resuspend the cells in BD Perm/Wash™ buffer at 10 x 10^6 /ml. 8. Aliquot test samples of 1 x 10^6 cells per 100- μ l test. 9. Add 20 μ l antibody per test, and incubate for 30 minutes at room temperature, protected from light. 10. Wash each test in 1.0 ml BD Perm/Wash™ Buffer, centrifuge and discard the supernatant. 11. Resuspend each test in 0.5 ml BD Perm/Wash™ Buffer and analyze by flow cytometry.