The explanted retinas were cultured in R16 medium with supplements for 2 days without treatment to adapt to the in vitro conditions. From P7 onwards, they were treated with different inhibitors: 0.1 μM SAHA (HY-10221; MedChemExpress, Sollentuna, Sweden), 1 μM Olaparib (HY-10162; MedChemExpress, Sollentuna, Sweden), and 20 μM Calpastatin (SCP0063; Sigma-Aldrich, St. Louis, MO, USA) for monotherapies and 0.1 μM SAHA and 20 μM CAST or 1 μM Olaparib and 20 μM CAST for combined therapies. There were at least five retinal explants from different animals in each group. During the culturing period, the R16 medium was changed every 2 days and the cultures were terminated at PN11 by fixation with 4% paraformaldehyde (PFA) or by direct freezing in liquid nitrogen (unfixed preparations). Afterwards, the retinas were embedded in O.C.TTM compound (4583; SAKURA, Alphen aan den Rijn, the Netherlands) and stored at −20 °C. The retina blocks were sectioned sagittally with 12 µm thickness using a microtome (Thermo Fisher Scientific, CryoStar NX50 OVP, Runcorn, UK) and collected on Superfrost Plus glass slides (R. Langenbrinck, Emmendingen, Germany). The glass slides were stored at −20 °C for further experiments.
Olaparib
Manufactured by Merck Group
Sourced in United States
Olaparib is a laboratory product manufactured by Merck Group. It is a poly(ADP-ribose) polymerase (PARP) inhibitor used in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Olaparib, by Selleck Chemicals (8 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (6 mentions)
Cisplatin, by Merck Group (5 mentions)
2 hydroxypropyl β cyclodextrin, by Merck Group (5 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (5 mentions)
Methylcellulose, by Merck Group (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
β actin, by Abcam (3 mentions)
Olaparib, by LC Laboratories (3 mentions)
Olaparib, by AstraZeneca (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Neurobasal a medium, by Thermo Fisher Scientific (3 mentions)
Olaparib, by MedChemExpress (2 mentions)
β tubulin, by Abcam (2 mentions)
Crystal violet, by Beyotime (2 mentions)
Cck 8 assay, by Beyotime (2 mentions)
Ripa assay, by Beyotime (2 mentions)
Loading buffer, by Beyotime (2 mentions)
Mitotempo, by Merck Group (2 mentions)
Stabilipan 2 x ray generator, by Siemens (2 mentions)
45 protocols using olaparib
1
Ex Vivo Retinal Explant Culturing
2
Evaluating Combination Therapy Potential
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Olaparib (PARP1 inhibitor, AZD2281), Rapa and CQ were from Sigma-Aldrich (St Louis, MO, USA). Icotinib (Ico) was provided by Betta Pharmaceutical Co., Ltd. (Hangzhou, China). Both Gef and Ico are first-line choice of EGFR sensitivity mutation and have similar clinical efficacy in NSCLC, however, compared with Gef, Ico has lower IC50 value, shorter half-life and lower adverse drug reactions. Antibodies against p62 and PARP1 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against mTOR, LC3, LC3II, β-tubulin and β-actin were from Abcam (Cambridge, MA, USA). CCK-8 assay, crystal violet, loading buffer, and RIPA assay were from Beyotime Institute of Biotechnology (Haimen, China).
3
Combination Treatment Effects on Cell Survival
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Target cells were plated in a six-well culture plate at 1 × 105 cells per well. The next day, the cells were treated with olaparib, mifepristone (Sigma-Aldrich), or both for the times indicated in the figure legends. Cell survival was determined by measuring propidium iodide (PI)–stained cells by flow cytometry as described previously (66 ). The DNA content of tumor cells was detected by PI staining and flow cytometry as described previously (67 ). For apoptosis assays, cells were incubated with the drugs for the times indicated in the figure legends and evaluated with a fluorescein isothiocyanate (FITC) Annexin V Apoptosis Detection Kit (556547, BD Biosciences). All flow cytometry experiments were conducted on a FACSCalibur flow cytometer (BD Biosciences). Data were analyzed with FlowJo software (Tree Star Inc.).
For immunofluorescence, cells or organoids were seeded onto a coverslip, treated as described in the figure legends, fixed, and stained with primary antibodies (table S6) overnight at 4°C. Samples were then incubated with a secondary antibody labeled with FITC (Invitrogen) at room temperature for 1 hour. After F-actin staining with phalloidin, samples were mounted with Vectashield mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories). Images were acquired with an Axio Imager A2 microscope.
For immunofluorescence, cells or organoids were seeded onto a coverslip, treated as described in the figure legends, fixed, and stained with primary antibodies (table S6) overnight at 4°C. Samples were then incubated with a secondary antibody labeled with FITC (Invitrogen) at room temperature for 1 hour. After F-actin staining with phalloidin, samples were mounted with Vectashield mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories). Images were acquired with an Axio Imager A2 microscope.
4
Comprehensive Chemical Sourcing for Research
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5
Investigating Olaparib and ROS Modulation
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Olaparib (O9201) and KU-55933 (K5050) were purchased from LC Laboratories (Woburn, MA, USA), dissolved in Dimethyl Sulfoxide (DMSO, Sigma-Aldrich) to 10 mM or 20 mM, respectively, and stored at -80°C. 5 μM Olaparib was used for in-vitro treatment unless otherwise indicated. Diphenyleneiodonium (DPI) and VAS-2870 were dissolved in DMSO, stored in -20°C, and used at 10 μM and 5 μM, respectively. Inhibitors were added to cells 1 hour before irradiation at desired concentrations. N-Acetyl-L-cysteine (NAC; Sigma-Aldrich, A9165) and MitoTEMPO (Sigma-Aldrich, SML0737) were dissolved in ddH2O and stored at -20°C. These compounds were aliquoted to avoid thaw-freeze cycles, with protection from light. ROS probes CM-H2DCFDA (DCF) and MitoSOX (Life Technologies) were dissolved in DMSO before each use to achieve concentrations of 10 mM and 5 mM, respectively. X-rays were produced by a Siemens Stabilipan 2 X-ray generator, which was operated at 250 kVp and 12 mA with a 2 mm Al filter, at a dose rate of 1.6 Gy/minute. Fresh human tumor samples were maintained in RPMI medium and treated within 2 hours of retrieval from patients with 10 μM Olaparib or 10 Gy X-rays as a control, followed by incubation for 24 hours, embedding in OCT (Sigma Aldrich), and snap freezing in liquid nitrogen for later analysis.
6
Combination Drug Treatment of BCLs
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BCLs were continuously treated for 72 h in adherent conditions with BO2 (RAD51i) (stock concentration SC = [10 mM], Sigma, SML0364), Cisplatin (SC = [10 mM], Selleckchem, S1166), Olaparib (PARPi) (SC = [10 mM], Selleckchem, S1060), PTC-209 (BMI1i) (SC = [10 mM], Selleckchem, S7372), 5-aza-2′-deoxycytidine (5-aza) (SC = [10 mM], Sigma, A3656), or APPO (SC = [10 mM], from R. Rodriguez’s laboratory, Institut curie, France) [40 (link)]. BO2, PTC-209, and Olaparib were resuspended in dimethyl sulfoxide (DMSO, Sigma), APPO was resuspended in DMF, and Cisplatin in PBS. For the in vivo experiments, BO2 (SC = [5 mg/ml]) was resuspended in a solution of DMSO/cremophor 20% PBS, PTC-209 (SC = [32 mg/ml]) was resuspended in a solution of DMSO/PEG400, and Cisplatin (SC = [150 mg/ml]) was resuspended in PBS.
7
Neuroprotective Compounds in C. elegans
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Nicotinamide riboside triflate (NR) was custom synthesized by Novalix (http://www.novalix-pharma.com/ ) and dissolved in water, and used at a final concentration of 1 mM. Olaparib (AZD2281) was purchased from Sigma Aldrich dissolved in DMSO to experimental concentrations of 300 nM. Thioflavin T (ThT) was purchased from Sigma-Aldrich (reference T3516-5G) dissolved in methanol and used at final concentrations of 5 and 50 uM (provided from L4 stage). Paraquat was purchased from Sigma-Aldrich (reference 856177), dissolved in water and used at final concentration of 4mM. Compounds were added just before pouring the plates. To ensure a permanent exposure to the compound, plates were changed twice a week.
8
Molecular Mechanisms of PARP1 Inhibition
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Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were from Invitrogen (Carlsbad, CA, USA). Olaparib (PARP1 inhibitor AZD2281), rapamycin (Rapa), hydroxychloroquine (CQ), penicillin, streptomycin and dimethyl sulfoxide (DMSO) were from Sigma‐Aldrich (St Louis, MO, USA). Antibodies against BDH1, p62 and PARP1 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against BDH1, mTOR, LC3, LC3II, β‐tubulin and β‐actin were from Abcam (Cambridge, MA, USA). COMET Assay kit (R&D Systems; Trevigen), CCK‐8 assay, crystal violet, loading buffer and RIPA assay were from Beyotime Institute of Biotechnology (Haimen, China).
9
MM Cell Line Characterization and Viability
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MM cell lines MM1S and NCI-H929 were obtained from American Type Culture Collection (ATCC; Manassas, VA). These cell lines were regularly authenticated using short tandem repeat polymorphism (STRP) analysis as recommended by ATCC, were mycoplasma free, and used within 6 months of receipt from ATCC. All in vitro studies were conducted in at least triplicate and in at least three independent experiments. Therapeutic reagents included melphalan, olaparib, and talazoparib (Sigma-Aldrich). Cell viability was assayed using the MTT Cell Proliferation Assay Kit (Roche, Indianapolis, IN) following the manufacturer's directions. Cellular apoptosis were measured using the Attune NxT Flow Cytometer and the standard manufacture recommended protocol (ThermoFisher). Further details are available in the Online Supplementary Appendix.
10
Synergistic Effects of Olaparib and DTIC on Melanoma
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Melanoma cells and NHEMs were plated at a density of 1 × 105 viable cells per well in a 6-well plates one day before drug treatment. Cells were cultured with 5 μM olaparib (Selleckchem), 2 mM dacarbazine (DTIC) (Sigma Aldrich), olaparib + DTIC, or vehicle. After 48 hours, half the cell suspension from each well was taken to determine cell viability after propidium iodide (PI) staining and cell cycle analysis. Following this, 1 ml of fresh medium containing drugs at appropriate concentrations was added to the remaining cell culture for additional 72 hours of culturing.
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