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.
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). Please refer to www.bdbiosciences.com/us/s/resources for technical protocols. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors. 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. Source of all serum proteins is from USDA inspected abattoirs located in the United States.