The GC cell lines NCI-N81, SNU16 and KATOIII were obtained from ATCC and ESO26 and OE19 were obtained from ECACC. All cell lines were cultured in RPM1-1640 media (Sigma) supplemented with 10% FBS and 1% Penicillin/Streptomycin, and maintained at 37 °C with 5% CO2. Cell line identity was confirmed by DNA fingerprinting (Source Biosciences). Cell lines were mycoplasma tested before and after in vitro experiments. Trastuzumab (21 mg/ml) was obtained from Beaumont Hospital pharmacy and was prepared in sterile water. Lapatinib (10.8 mM) was purchased from Selleckchem and stock solutions were prepared in dimethylsulfoxide (DMSO). The PI3K inhibitor copanlisib and the MEK1/2 inhibitor refametinib were obtained from Selleckchem and stocks (5 mM copanlisib; 10 mM refametinib) were prepared in 100% DMSO with 10 mM TFA and 100% DMSO, respectively.
Copanlisib
Manufactured by Selleck Chemicals
Sourced in United States, China
Copanlisib is a laboratory reagent used for research purposes. It is a PI3K inhibitor that targets the alpha and delta isoforms of the phosphoinositide 3-kinase (PI3K) enzyme. Copanlisib is utilized in various research applications to investigate cellular signaling pathways and potential therapeutic targets.
Automatically generated - may contain errors
Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Vincristine, by Selleck Chemicals (3 mentions)
Refametinib, by Selleck Chemicals (2 mentions)
Lapatinib, by Selleck Chemicals (2 mentions)
Celltiter 96 aqueous one solution cell proliferation assay, by Promega (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Afatinib, by Selleck Chemicals (2 mentions)
D2650, by Merck Group (2 mentions)
Bafilomycin a, by Merck Group (2 mentions)
Copanlisib, by Merck Group (2 mentions)
Pictilisib, by Selleck Chemicals (2 mentions)
Ngi 1, by Selleck Chemicals (2 mentions)
Duvelisib, by Selleck Chemicals (2 mentions)
Rpm1 1640 media, by Merck Group (1 mentions)
Vorinostat, by Selleck Chemicals (1 mentions)
Erlotinib, by Selleck Chemicals (1 mentions)
Prism 8, by GraphPad (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
18 protocols using copanlisib
1
GC Cell Culture and Treatment Protocols
2
Investigating Combination Therapies for Hypopharyngeal and Laryngeal Cancers
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The human hypopharyngeal cancer cell line, FaDu, was obtained from the American Type Culture Collection (ATCC), American. The human laryngeal cancer cell line, TU212, was acquired from Beijing Zhongke Quality Inspection Biotechnology Co. Ltd. (Beijing, China). The cells were cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS). Erlotinib (E), vorinostat (V), and copanlisib (C) were provided by Selleck Inc. (Shanghai, China), and diluted in 10% FBS RPMI 1640 medium at working concentrations (Erlotinib: 10 µM; vorinostat: 1 µM; copanlisib: 90, 120 nM) for a 72 h combination drug intervention. The treatment groups were designated as follows: (I) Erlotinib + vorinostat + copanlisib; (II) Erlotinib + vorinostat; (III) Erlotinib + copanlisib; (IV) Erlotinib; (V) vorinostat + copanlisib; (VI) vorinostat; (VII) copanlisib; and (VIII) dimethyl sulfoxide (DMSO).
3
Investigating Targeted Inhibitors in Cancer
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Cell lines were mycoplasma tested before and after the in vitro experiments. The Shp2 inhibitor (SHPi) (SHP099), PI3K inhibitor copanlisib (cop) and the MEK1/2 inhibitor refametinib (ref) were obtained from Selleckchem and stocks (10 mM SHPi, 10 mM refametinib, 5 mM copanlisib;) were prepared in 100% DMSO, 100% DMSO and 100% DMSO with 10 mM TFA, respectively. The growth inhibition of Ba/F3 and NSCLC cells was assessed by a CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA) and acid phosphatase assays, respectively. The data were graphically displayed using GraphPad Prism version 6.00 for Windows (GraphPad Software). The curves were fitted using a nonlinear regression model with a sigmoidal dose-response. The cellular viability of PTPN11 WT and mutated cells was assessed by the Alamar Blue assay, and fluorescence was measured at 610 nm using VICTORTM X3 2030 Multilabel Plate Reader. Statistical analysis was performed using Graphpad Prism 8.4.3. Statistical significance was calculated following the software-recommended tests and post-tests and is described in individual figure legends.
4
Comprehensive Reagent and Cell Line Protocol
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Afatinib, dasatinib, pazopanib, amuvatinib, crizotinib, sunitinib, sorafenib tosylate and copanlisib were purchased from Selleckchem (Houston, TX). Trypsin-EDTA, DMEM, RPMI, penicillin-streptomycin were purchased from GIBCOBRL (GIBCOBRL Life Technologies, Grand Island, NY). Cells were purchased from the ATCC and were not further validated beyond that claimed by ATCC. Cells were re-purchased every ∼6 months. Commercially available validated short hairpin RNA molecules to knock down RNA / protein levels were from Qiagen (Valencia, CA) or were supplied by collaborators. Reagents and performance of experimental procedures were described in [15 (link)–18 (link)].
5
Cell Viability Assay for Drug Combination
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Cell viabilities were measured by Cell Counting Kit-8 (CCK-8) (#96992, Millipore Sigma) following manufacturer’s protocol. In brief, cells were plated at 1×105 cells/well in 96-well plates and incubated at 37°C and 5% CO2 for 4 hours before treatment with serial concentrations of copanlisib (#S2802, Selleck Chemicals), domatinostat (#S7555, Selleck Chemicals), or in combination for 72 hours. CCK-8 reagent (10% of well volume) was added to each well and incubated for 2-4hours at 37°C before measuring optical density (OD) at 450nm using a spectrophotometer. Half-maximal growth inhibitory dose (GI 50) was calculated by plotting dose-response curve, normalized against vehicle-treated cells, using GraphPad Prism.
6
Spheroid Formation Assay and Analysis
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The spheroid formation assay was performed as previously described.13 The spheroid‐forming efficiency was defined as the ratio between number of spheroids and number of cells seeded. To perform the whole‐exome sequencing and the mRNA sequencing, first‐generation spheroids were dissociated and re‐seeded as described above to obtain second‐generation spheroids. When needed, the PI3K chemical inhibitors LY294002 (Cell Signaling Technology, Danvers, MA), Alpelisib (BYL‐719; Selleckchem, Houston, TX) or Copanlisib (BAY 80‐6946; Selleckchem) were added at the indicated concentrations to suspension cultures on days 0, 2 and 4 after seeding. Spheroid formation was assessed 7 days after seeding. See also Supporting Information for further information.
7
Pharmacological Inhibition of Key Pathways
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Pharmacological inhibitors of MEK1/2 (Trametinib/GSK1120212), PI3K (Copanlisib/BAY 80-6946), and DNMT1 (GSK3685032) were acquired from Selleckchem. KRAS G12C inhibitor (Sotorasib/AMG-510) was purchased from MedChemExpress.
8
Evaluation of ZSTK474, Copanlisib, and Z-VAD-FMK
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ZSTK474 was provided by OHARA Pharmaceutical Co., Ltd. (Tokyo, Japan). Copanlisib was purchased from Selleck Chemicals (Houston, TX) and Z-VAD-FMK was purchased from Adipogen Life Sciences (Liestal, Switzerland). These compounds were dissolved in dimethyl sulfoxide.
9
Anticancer Drug Combination Protocol
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The followings drugs were used: Vincristine and Copanlisib (BAY 80–6946) were obtained from Selleckchem (Houston, TX, USA) and Prednisolone (P6004) obtained from Merck. All drugs were prepared at the appropriate stocking concentrations in DMSO (Merck) and stored at −20 °C until use.
10
Culturing HD-MBO3 Medulloblastoma Cells
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HD-MBO3 Group 3 medulloblastoma cells were cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS, both from Sigma-Aldrich, St. Louis, USA), 1% Penicillin-Streptomycin, and 1% GlutaMAX (both from Gibco/Thermo Fisher Scientific, Waltham, USA). The cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2. Chemicals: Copanlisib (BAY 80-6946, Selleckchem, Munich, Germany) and other compounds were dissolved in 100% dimethyl sulfoxide (DMSO) at a stock concentration of 10 to 100 mM and stored at −20 °C. DMSO was used as solvent control in all assays.
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