BD, 552933, BD Pharmingen™ PE Mouse Anti-Cleaved PARP (Asp214)

Reactivity: Human (QC Testing)
Isotype: Mouse IgG1, κ
Immunogen: Human cleaved PARP
Application: Intracellular staining (flow cytometry) (Routinely Tested)
Vol. Per Test: 20 µl
RRID: AB_647224
Storage Buffer: Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
Regulatory Status: RUO
Preparation And Storage: Preparation And Storage The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated with R-PE under optimum conditions, and unconjugated antibody and free PE were removed. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
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 µM 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 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 mM 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 hours at 37°C. 3.  Wash the cells (camptothecin-treated and control aliquots) twice with cold PBS; then resuspend them in 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 20 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.