(C) Aliquots containing 50 g of whole cell lysate from?the P388/CPT and parental P388 mouse lymphoma lines?(lanes 1 and 2) or serial 2-fold dilutions (lanes 3-5) were subjected to SDS-PAGE, probed for topo I and, as a loading control, poly(ADP-ribose) polymerase 1 (PARP1). applied to study additional topoisomerases and their inhibitors and complexing of enzyme (ICE) assays (36) and potassium-SDS precipitation assays (37C39). Alkaline elution, which separates nicked from intact DNA by filtration, is time-consuming, needs specialized equipment and typically requires high drug concentrations ( 250 nM TPT) to detect covalent topo I-DNA complexes. ICE assays, which involve cell lysis followed by Niraparib R-enantiomer ultracentrifugation to separate covalent topo I-DNA complexes from free protein, are lengthy (20+ h for ultracentrifugation alone) and even less sensitive. Potassium-SDS methods, which involve precipitation of proteins along with any covalently bound DNA, are not specific for topo I-DNA covalent complexes and usually require radiolabeling of DNA as Niraparib R-enantiomer well as reproducible DNA shearing for sensitive, accurate quantitation. A more recently described method that uses chaotropic salts to rapidly denature protein and recover DNA-bound protein (40) has improved sensitivity for topo I-DNA covalent complexes but is limited to immunoblot- or ELISA-based detection and cannot be paired with immunofluorescence or flow cytometry. To overcome these difficulties, we have developed a monoclonal antibody with specificity for topo I covalently bound to DNA that is capable of detecting topo I-DNA covalent complexes by immunoblotting, immunofluorescence or flow cytometry. Here, we utilize this antibody to detect topo I-DNA covalent complexes and for 5 min, decanted and fixed by incubation in 2% paraformaldehyde in PBS for 15 min at 4C. After sedimentation at 150for 5 min, cells were treated with 0.25% (w/v) Triton X-100 in PBS (15 min, 4C), sedimented at 150for 5 min, resuspended in PBS and immediately subjected to flow microfluorimetry on a MAPT FACSCanto II flow cytometer (BD Biosciences; San Jose, CA, USA) using the FL4 channel (excitation: 633 nm; emission: 660/20 nm). Data were analyzed and overlays created using BD CellQuest software. Xenografts Once subcutaneous xenografts of A549 cells in nu/nu mice (Harlan labs) reached 0.7 Niraparib R-enantiomer cm in their longest axis, mice were treated with 50 mg/kg irinotecan intraperitoneally. Tumors harvested before or 2-6 h after treatment were snap frozen in liquid nitrogen, embedded Niraparib R-enantiomer in OTC embedding medium, stored at -80C, sectioned onto -20C slides and immediately fixed and prepared for immunostaining using conditions described above for tissue culture cells. RESULTS To test the hypothesis that topo I covalently attached to DNA could be selectively detected immunologically, monoclonal antibodies were raised against a peptide corresponding to the active site of the topo I with a phosphorylated Tyr723 residue (Figure ?(Figure1B).1B). Of 726 wells screened, one hybridoma was found to react with the immunizing peptide in ELISA assays and preferentially recognize topo I-DNA complexes by immunoblotting upon secondary screening (Supplementary Figure S1). That antibody, termed -TopoIcc, was purified from hybridoma supernatants and used for further studies. Detection via immunoblotting Slot blotting demonstrated that -TopoIcc detects topo I-DNA covalent complexes in cell Niraparib R-enantiomer lysates with high specificity. Following treatment with topoisomerase poisons, cell lysates were prepared under denaturing conditions and subjected to cesium chloride centrifugation to separate DNA-bound and free proteins. Upon subsequent immunoblotting, -TopoIcc detected topo I that co-migrated with DNA in lysates from TPT-treated cells (Figure ?(Figure2A).2A). Importantly, -TopoIcc did not detect topo II-DNA.