HeLa WT and PARP1 KO cells were cultured at 37°C, 5% CO2 and 95% humidity in DMEM (Gibco) supplemented with 10% fetal bovine serum (Biochrom), 90 U/ml penicillin, 90 μg/ml streptomycin (Gibco) and 2 mM glutamine (Gibco). For transient transfections with the PARP1-eGFP constructs, Effectene transfection reagent (Qiagen) was used according to the manufacturer's instruction, except for the amount of Effectene, which was reduced by 50%. Treatment with 10 μM ABT888 (Selleckchem) was performed immediately before transfection and cells were incubated with ABT888 until harvesting. To determine transfection efficiencies, transfected HeLa PARP1 KO cells were harvested with trypsin-EDTA (Gibco), pelleted by centrifugation, and resuspended either in phenolred-free medium or in FACS-buffer [PBS, 0.5 mM EDTA, 1% (v/v) BSA]. Samples were analyzed using FACSCalibur (BD), FACSFortessa (BD) or FACSVerse (BD) instruments, depending on the experiment.
Abt 888
Manufactured by Selleck Chemicals
Sourced in United States, Germany
ABT-888 is a small-molecule inhibitor of Poly(ADP-ribose) polymerase (PARP) enzymes. It is commonly used in laboratory research applications involving the study of PARP-related biological processes and pathways.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (5 mentions)
Streptomycin, by Thermo Fisher Scientific (5 mentions)
Gibson assembly cloning kit, by New England Biolabs (2 mentions)
Cisplatin, by Merck Group (2 mentions)
Azd2281, by Selleck Chemicals (2 mentions)
Dacarbazine, by Merck Group (2 mentions)
Rpmi f 12, by Thermo Fisher Scientific (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
H727 cells, by American Type Culture Collection (2 mentions)
Olaparib, by Selleck Chemicals (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Mouse alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Melphalan, by Merck Group (1 mentions)
Decitabine, by Johnson & Johnson (1 mentions)
Bleomycin, by Santa Cruz Biotechnology (1 mentions)
Bleomycin, by Selleck Chemicals (1 mentions)
Senescence β galactosidase staining kit, by Cell Signaling Technology (1 mentions)
Z vad fmk, by Apexbio (1 mentions)
24 protocols using abt 888
1
PARP1 KO Cell Culture and Transfection
2
Herpes Simplex Virus Transduction of Spinal Cord Neurons
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Mixed spinal cord cultures were prepared from rat and transfected with virus following previously established protocols [77 (link), 78 (link)]. The titer of herpes simplex virus routinely used in our studies was 3-5 × 107 plaque forming units (pfu)/ml [78 (link)]. The primary neuron cultures were infected 14 days in vitro (DIV) with herpes simplex virus (HSV) expressing either TDP-43 or LacZ. The inhibitor Veliparib, also called ABT-888 (Selleckchem, # S1004), or DMSO was added to the cell-culture medium at the indicated concentration at the time of infection. Media was changed 3-days post infection and cells were fixed and processed for immunofluorescence on day 5 of infection. Mouse anti-neurofilament-H, NF-H (1 in 1000, Biolegend #801703) and mouse AlexaFluor 488 (1 in 500, ThermoFisher, # A-21203) were used to identify neurons. Five images (10X magnification) were captured from each condition and remaining neuronal cell bodies were counted. Each condition was repeated three times, on three independent cultures. Statistics were performed in Graphpad 6 software.
3
Evaluation of Decitabine and Quisinostat Combination
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Decitabine (Dacogen) and JNJ-26481585 (quisinostat; JNJ-585) were kindly provided by Johnson & Johnson (Beerse, Belgium). Melphalan and B02 were obtained at Sigma. ABT-888 was obtained from Selleckchem (Munich, Germany). Decitabine, JNJ-585, ABT-888 and B02 were dissolved in dimethylsulfoxide. Melphalan was dissolved in acidified ethanol. For in vivo experiments, Decitabine and JNJ-585 were used as a filter sterilized 10% hydroxypropyl-cyclodextran suspension.
4
Assay for Base Excision Repair Activity
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A 30 μL aliquot of extract was added to each minigel in the incubation chamber. Each extract was incubated with Ro-treated as well as non-treated PBMC (used for background subtraction). Incubation time was 20 min, at 37°C in a humid environment. Fpg was used as a positive control. For a negative control substrate DNA was incubated with buffer A + buffer B in a 1:4 ratio. Each experimental point was performed in duplicate. In optimization experiments, PARP inhibitor ABT-888 (Selleckchem) was added to the extract at a concentration of 5 μM to test the effect of inhibiting the post-incision phase of BER.
5
Generation and Validation of Brca1 Mutants
6
BRCA1 Mutation and Repair Assay
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MMC (SCBT, Santa Cruz, CA, USA), cisplatin (Sigma-Aldrich, St Louis, MO, USA), ABT-888 (SelleckChem, Houston, TX, USA) and bleomycin (SCBT) were purchased in dimethyl sulfoxide or prepared as stocks in dimethyl sulfoxide. The Brca1 deletion mutants36 (link) are HA tagged and correspond to deletions within the Brca1 protein as illustrated in Figure 6d . The Brca1 ΔM3 mutant (amino acids 1290–1530) was generated from full-length HA-Brca1 plasmid37 using methods described in Gibson Assembly Cloning Kit (New England BioLabs, Ipswich, MA, USA) and primer sequences: BRCA1-305-For, 5′-CAGAATGAATGTAGAAAAGGCTG-3′ and BRCA1-305-1290-del-Rev, 5′-GTTGCTCCTCCACATCAACAACACATTTTGTTTCCTCACTAAG-3′; BRCA1-305-1290-del-For, 5′-CTTAGTGAGGAAACAAAATGTGTTGTTGATGTGGAGGAGCAAC-3′ and pcDNA-Xho-rev, 5′-TAGGGCCCTCTAGATGCATGC-3′. XhoI and EcoRI restriction digestion and sequencing validated the correct insertion and sequence. The full-length myc-Wwox plasmid13 (link) and deletion mutants15 (link) were described previously.
7
Senescence Induction and Inhibition
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Salubrinal was from Calbiochem-Merck4Biosciences (Darmstadt, Germany), vorinostat from LC laboratories (Boston, MA), ABT-888 from SelleckChem or APExBIO (Houston, TX), 6-TG from Tocris bioscience (Bristol, UK) or Sigma, (St. Louis, MO.), Z-VAD-FMK was purchased from APExBIO and senescence β-galactosidase staining kit was purchased from Cell Signaling Technologies (Boston, MA).
8
Dual PARP Inhibitor Evaluation
9
Bicalutamide and ABT-888 Synergistic Effects
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Bicalutamide and ABT-888 with a purity of 99% were purchased from Selleck Chemicals. Dihydrotestosterone (DHT), 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were from Sigma Chemicals. DHT was dissolved in ethanol at 0.1μM concentration, Bicalutamide and ABT-888 were dissolved in DMSO at 10 μM concentration. Cell Apoptosis Detection Kit was a product of KeyGEN Biotech, CA. The antibodies used included the following: AR (Abcam, ab74272), PARP1 (Abcam, ab32138). All culture media and serum were obtained from GIBCO/BRL Life Technologies, Inc.
10
Synergistic Effects of ABT-888 and Dacarbazine on Carcinoid Cells
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Human gastrointestinal carcinoid cells (BON), were gifted by Drs. Courtney M. Townsend, Jr. of the University of Texas Medical Branch (Galveston, TX, USA) and B. Mark Evers of the University of Kentucky (Lexington, KY, USA). Human bronchopulmonary carcinoid (H727) cells were purchased from the American Type Culture Collection (Manassas, VA, USA). BON and H727 cells were grown in DMEM/F-12 (Life Technologies, Grand Island, NY, USA) and RPMI/F-12 (Life Technologies, Grand Island, NY, USA), respectively, at a 5% CO2 and 37°C atmosphere. Media was supplemented with 100 IU/mL penicillin, 100µg/mL streptomycin (Life Technologies, Grand Island, NY, USA) and 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA). ABT-888 (Selleck Chemicals, Houston, TX, USA) and dacarbazine (Sigma-Aldrich) were stored in aliquots of 10mM in DMSO at -80°C, and freshly thawed before use. Cells were plated at sub-confluency the day prior to treatment, and then incubated in fresh medium containing ABT-888 (0–10µM) for 24 hours, after which dacarbazine was added (0–1000µM) for 2 additional days. DMSO concentrations were normalized across all treatment groups.
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