TTGM 5826, and other small molecules tested against tTG, were obtained from ChemBridge (San Diego, CA). GSCs were obtained as previously described [60 (link), 61 (link)]. All other cell lines used in the study were purchased from the ATCC (Manassas, VA). RPMI 1640, DMEM, DMEM-F12, Penicillin/Streptomycin, Fungizone, B27 supplement, heparin, bFGF, EGF, Trip-LE, trypsin-EDTA, bodipy-GTP-γS, horse serum (HS), and fetal bovine serum (FBS) were purchased from Invitrogen (Waltham, MA). MEGM was obtained from Lonza (Allendale, NJ). Recombinant tTG was prepared as previously described [51 (link)]. HRP-conjugated streptavidin and BPA were obtained from Pierce Biotechnology (Waltham, MA), and the human-specific tTG antibody (MS-300-P, used for human-derived cancer cells) was from Neomarkers (Fremont, CA). The mouse-specific TG antibody (A033, used for MEFs) and Z-Don were from Zedira GMBH (Darmstadt, Germany). The anti-rabbit IgG-HRP (7074S), anti-mouse IgG-HRP (7076S), and HA antibodies (3724S) were from Cell Signaling (Danvers, MA). The vinculin antibody (V9131), purified trypsin, soybean trypsin inhibitor, temozolomide, vincristine, carmustine, MDC, DMSO, and NNDC were obtained from Sigma Aldrich (St. Louis, MO).
Carmustine
Manufactured by Merck Group
Sourced in United States
Carmustine is a cytotoxic agent used in the treatment of various types of cancer. It is a laboratory product that functions as an alkylating agent, interfering with DNA synthesis and cell division. Carmustine is commonly used in research and clinical settings to study its effects on cancer cells.
Automatically generated - may contain errors
Lab products found in correlation
Temozolomide, by Merck Group (6 mentions)
Cisplatin, by Merck Group (4 mentions)
Etoposide, by Merck Group (3 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (2 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (2 mentions)
Vincristine, by Merck Group (2 mentions)
Dacarbazine, by Merck Group (2 mentions)
Silver nitrate, by Merck Group (2 mentions)
Amsacrine hydrochloride, by Thermo Fisher Scientific (2 mentions)
Bortezomib, by Avantor (2 mentions)
Puromycin dihydrochloride, by Avantor (2 mentions)
Bleomycin sulfate, by Thermo Fisher Scientific (2 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated streptavidin, by Thermo Fisher Scientific (1 mentions)
Soybean trypsin inhibitor, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
18 protocols using carmustine
1
Transglutaminase Inhibitors and Glioblastoma
2
Detailed Reagent Acquisition Protocol
3
Characterization of Redox-Sensitive Proteins
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The plasmid vector encoding V5‐tagged α1‐antitrypsin, influenza virus HA, bovine preprolactin, and cVIMP‐cys were gifted from Lisa Swanton (University of Manchester), Mary Jane Gething (University of Melbourne), Stephen High (University of Manchester), and Lars Ellgaard (University of Copenhagen), respectively. The β1‐integrin, hemagglutinin, and the soluble Sec‐Cys variant of SelS/VIMP (cVIMP‐cys) sequences have been described previously (Gething et al, 1980 ; Tiwari et al, 2011 ; Christensen et al, 2012 ). Auranofin, carmustine, and DHEA were purchased from Sigma‐Aldrich; TRi2 synthesis will be described elsewhere (Stafford, W. et al, manuscript in preparation). The commercial antibodies used were anti‐His (Proteintech), anti‐V5‐tag (Invitrogen), and 9EG7 (BD Bioscience). Antibody to LDLr (121) was a gift from Ineke Braakman (Utrecht University; Pena et al, 2010 ). Purified recombinant ChaC1 enzyme was a gift from David Ron (University of Cambridge; Tsunoda et al, 2014 ). The plasmid containing human thioredoxin (hTrx) with a streptavidin binding peptide (SBP) tag was a gift from Tobias Dick (German Cancer Research Centre, Heidelberg). Recombinant Trx protein was purified as described previously (Schwertassek et al, 2007 ).
4
Comprehensive Chemotherapeutic Reagent Procurement
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The following reagents were purchased from Sigma: MG-132, chlorambucil, cyclophosphamide, carmustine, busulfan, dacarbazine, thiotepa, cisplatin, 5-fluorouracil, 6-mercaptopurine, gemcitabine, methotrexate, doxorubicin, epirubicin, actinomycin-D, mitomycin-C, topotecan, irinotecan, etoposide, mitoxantrone, paclitaxel, docetaxel, and vincristine. The following reagents were purchased from Calbiochem: carboplatin, pentostatin, and daunorubicin. The following reagents were purchased from Santa Cruz: ICRF-187, ICRF-193, and teniposide. The following reagents were purchased from MBL International Corporation: Z-VAD-FMK.
5
Characterization of GBM Cell Lines
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FN and carmustine (both from Sigma–Aldrich, St. Louis, MI, USA) stock solutions were made by dissolving these at 2 mg/mL in phosphate buffered saline (PBS) without Ca2+ or Mg2+. These were aliquoted and stored at −80°C. Basic fibroblast growth factor (b-FGF) and epidermal growth factor (EGF; both from PeproTech, Rehovot, Israel) were diluted in 0.9% NaCl. Cilengitide was purchased from Life Technologies (Carlsbad, CA, USA). For immunofluorescence staining, the following antibodies were bought: nestin (Thermo Fisher Scientific, Waltham, MA, USA), glial fibrillary acidic protein (GFAP; Aves Lab, Tigard, OR, USA), β-tubulin (Abcam, Cambridge, UK), Ki67 and sox-2 (Millipore, Billerica, MA, USA) and isotype control Ig1 (Cell Signaling Technology, Danvers, MA, USA). Primers for quantitative polymerase chain reaction (qPCR) of sox-2, β-tubulin and GAPDH were obtained from Life Technologies and primers for GFAP were from Thermo Fisher Scientific. Antibodies against GAPDH and secondary antibodies were purchased from Thermo Fisher Scientific. Primary antibodies against matrix metallopeptidase (MMP)-2/-9, t-/p-Focal adhesion kinase (FAK), p-paxillin, t-/p-AKT, P-glycoprotein, p-ERK1/2 and cyclin D1 for western blotting were purchased from Cell Signaling Technology (Danvers, MA, USA).
6
Cytotoxic Agents and Cancer Cell Treatments
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HAT supplement (50X) was from Sigma. Dacarbazine, carmustine, cisplatin and temozolomide and lomeguatrib were from SIGMA. Hypoxanthine, guanosine and 5′-guanosine monophosphate were from Sigma. AZD6244 was from Selleck Chemicals, Newmarket, UK. Mycophenolic Acid and AVN944 were from Sigma and ChemieTek respectively. Aminopterin, pyrimethamine and amethopterin (methotrexate) were from Sigma. All drugs were dissolved in dimethylsulfoxide (DMSO) and, apart from Dacarbazine, directly added to cell in culture at the indicated concentrations. Prior to addition onto cells DTIC was exposed to white light for 1 h, as previously described [28 (link),30 (link)].
7
Western Blot Antibody Validation Protocol
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Lomustine, carmustine, oxaliplatin, UCN-01, temozolomide, tamoxifen and GAPDH antibody were purchased from Sigma (USA). Antibody to GRP78 was from Abcam and Santa Cruz Biotechnology (USA). Bcl-2, ERK42/44 and Par-4 antibodies were obtained from BD Bioscience, Santa Cruz Biotechnology (USA), Sigma and Cell Signaling Technology (CST). Phospho Akt (Ser473) and total Akt antibodies were purchased from Santa Cruz Biotechnology (USA). Species specific HRP-labeled and fluorescent labeled secondary antibodies were procured from Bio-rad (USA) and Molecular probes (USA) respectively.
8
Chemoresponse Assessment via Modified Clonogenic Assay
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For chemoresponse assessment, a modification of clonogenic assay as described by Hamburger and Salmon was used (8 (link)). The bottom layer consisted of 0.2 ml/well DMEM supplemented with 20% FBS and 0.75% agar; 20 × 104 cells obtained by primary culture or direct single-cell suspension were added to 0.2 ml of the medium containing 0.4% agar and plated in 24-multiwell dishes on top of the bottom layer. Temozolomide and carmustine (Sigma-Aldrich, USA) were added as drug overlays in 0.2 ml medium containing 1 to 100 µM of drugs in triplicate. Cultures were incubated at 37°C under 5% CO2 in a humidified atmosphere for up to 21 days and monitored closely for colony formation using an inverted microscope. In day 21, counting of colonies with more than 50 µm in diameter was performed, and surviving fraction was calculated by colony count of treated group divided by colony formation of control group. Finally, median inhibitory concentration (IC50) was determined as the drug concentration that is required to reduce the colony formation to half that of the control.
9
Cell Proliferation Assay for Glioma Stem Cells
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Cell proliferation assays were carried out using a Cell Titer 96™ tetrazolium compound (MTS) kit according to the manufacturer’s instructions. Briefly, 10,000 GSCs/well were pre-incubated in 96-well plates coated with 0, 1, 5, or 10 μg/mL FN for 24 h. Cells were then treated with 200 μM carmustine (Sigma–Aldrich, dissolved in 100% ethanol) or 1/1000 diluted ethanol (as control). Cells either formed spheres or adhered onto FN-coated wells after treatment for 72 h, after which 10 μL MTS solution was added to each well. A microplate reader (BioTek, USA) was used to measure the optical density (OD) of each well at 490 nm after incubating plates for another 2 h. Experiments were repeated three times with five replicates per experiment.
10
High-throughput drug screening in C. elegans
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Four genetically divergent strains (N2, CB4856, JU258, and DL238) were treated with increasing concentrations of each of the eight drugs using the high-throughput fitness assay described above. The dose of each drug that provided a reproducible drug-specific effect that maximizes between-strain variation while minimizing within-strain variation across the four traits was selected for the linkage mapping experiments. The chosen concentrations are as follows: 100 µM amsacrine hydrochloride (Fisher Scientific, #A277720MG) in DMSO, 50 µM bleomycin sulfate (Fisher, #50-148-546) in water, 2 µM bortezomib (VWR, #AAJ60378-MA) in DMSO, 250 µM carmustine (Sigma, #1096724-75MG) in DMSO, 500 µM cisplatin (Sigma, #479306-1G) in K media, 500 µM etoposide (Sigma, #E1383) in DMSO, 500 µM puromycin dihydrochloride (VWR, #62111-170) in water, and 150 µM silver nitrate (Sigma-Aldrich, #S6506-5G) in water.
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