SN-38 (Sigma-Aldrich, Copenhagen, Denmark) was purchased and dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mM and stored at -20 °C. The indenoisoquinoline drugs NSC 725776 (LMP776) and NSC 743400 (LMP400), provided by the laboratory of Dr. Yves Pommier, were dissolved in DMSO at a concentration of 5 mM and stored at -20 °C. Epirubicin (2 mg/ml, Actavis Nordic A/S, Gentofte, Denmark) and etoposide (20 mg/ml, Pfizer, New York, USA) were purchased and stored at -20 °C. Drugs were diluted in growth medium immediately prior to use.
Etoposide
Manufactured by Pfizer
Sourced in Australia
Etoposide is a laboratory product used as a standard reference material in analytical testing and research applications. It is a chemical compound that can be used as a control or calibrator in various analytical procedures, such as high-performance liquid chromatography (HPLC) or mass spectrometry, to ensure the accuracy and reliability of test results.
Automatically generated - may contain errors
Lab products found in correlation
2 mercaptoethanol, by Merck Group (2 mentions)
Asparagine, by Merck Group (2 mentions)
Sn 38, by Merck Group (1 mentions)
Platinol, by Bristol-Myers Squibb (1 mentions)
Adriamycin, by Pfizer (1 mentions)
Ly2109761, by Selleck Chemicals (1 mentions)
Facscalibur, by BD (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Pdgf bb, by Merck Group (1 mentions)
Topotecan, by GlaxoSmithKline (1 mentions)
Anti gadd45α, by Cell Signaling Technology (1 mentions)
Cisplatin, by Bristol-Myers Squibb (1 mentions)
Carboplatin, by Bristol-Myers Squibb (1 mentions)
Docetaxel, by Bristol-Myers Squibb (1 mentions)
Perm wash buffer, by BD (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Bovogen (1 mentions)
6 protocols using etoposide
1
Preparation of Anticancer Drug Solutions
2
Chemotherapeutic Agents Procurement Protocol
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Methotrexate was purchased from Medac Pharma, Inc., USA. Etoposide was purchased from Pfizer Inc., USA. Ifosfamide (Ifex®) was purchased from Bristol-Myers Squibb Co. New York, USA. Doxorubicin (Adriamycin®) was purchased from Pfizer, Inc., USA. Cisplatin (Platinol®) was purchased from Bristol-Myers Squibb Co., New York, USA. Zoledronate (Zometa®) was purchased from Beijing Novartis Pharma Co., Beijing, China. Calcium and 25-Hydroxyvitamin D tablets were purchased from Glenmark Pharmaceuticals, India. Normal saline was purchased from Shanghai Baxter Healthcare Co., Shanghai, China.
3
Quantitative Analysis of DDR and TGFβ-Induced Signaling
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For Western blot and standard immunocytochemistry assays cells were grown as monolayer as described in [15 (link)]. To test DDR, cells were grown in the growth media supplemented with 0.05–2 µM etoposide (Pfizer #391870) for 5 h–3 days as indicated. For experimental series containing 3.3–50 pM TGFβ (Abcam #50038) and/or 1 µM LY2109761 (Selleckchem #S2704) -treatments cells were grown in the growth media with reduced EGF concentration (2 ng/ml) for 24 or 48 h as indicated. The final concentration of DMSO vehicle in the experimental media for LY2109761 treatment was 0.0025%. Spheroids were grown on top of GFR-BME-coated chamber slides for 8 days and stained as described [15 (link), 16 (link)]. Proteins were quantified in Western blot analyses with total protein normalization. Protocols, antibodies and stains have been provided in detail in Supplementary Methods. Validation of PALB2 antibodies has been demonstrated in Supplementary Fig. 2a.
4
Quantifying Thymocyte Apoptosis Induction
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CD4+CD8+ DP thymocytes were sorted using a MoFlo (Cytomation, Fort Collins, CO, USA) high-speed sorter and then plated in 100 μl of high glucose Dulbecco's modified Eagle's media (DME Kelso) supplemented with 10−6 M asparagine (Sigma-Aldrich, St. Louis, MO, USA), 50 μM 2-mercaptoethanol (Sigma-Aldrich) and 10% FCS (Gibco, Mulgrave, VIC, Australia) at a concentration of 50 × 103 cells per well with or without stimulus at a final concentration of 32 ng/ml Fc-FasL (produced at WEHI using plasmid ps1117 kindly provided by Dr Pascal Schneider, University of Lausanne) or 1 μg/ml etoposide (Pfizer, West Ryde, NSW, Australia). Cell viability was measured using a FACSCalibur (BD Biosciences) 0, 4, 12, 24 and 48 h after treatment with a cytotoxic stimulus by staining with annexin V-Alexa647 (in-house) and propidium iodide (PI; Sigma-Aldrich). Viability (live cells identified as annexin V–PI–) was calculated relative to untreated samples to determine the percentage of stimulus-induced apoptosis and then normalized to viability at 0 h.
5
Chemotherapeutic Compounds Protocol
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The chemotherapy compounds mitoxantrone (MX) (Wyeth-Lederle, Finland), doxorubicin (DXR) (Nycomed, Roskilde, Denmark), cyclophosphamide (Orion Pharma, Espoo, Finland), topotecan (GlaxoSmithKline, Uxbridge, Middlesex, UK), etoposide (Pfizer), cisplatin (Bristol-Myers Squibb, Princeton, NJ, USA), docetaxel (Aventis), and carboplatin (Bristol-Myers Squibb, Princeton, NJ, USA) were stored and prepared as described [17] (link). Imatinib was a gift from Novartis Pharmaceuticals (Basel, Switzerland). The stock solution of imatinib at 200 mM was prepared by dissolving the compound in DMSO. The c-kit and PDGF-α and β receptor blocker AG1296 was purchased from Calbiochem (cat. no. 658551). PDGF-BB was purchased from Sigma-Aldrich. The following antibodies were used for Western blotting: mouse monoclonal DO-1 for p53 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit polyclonal anti-GADD45α (Cell Signaling, cat. no. 3518), rabbit polyclonal anti-phospho-PDGF β receptor (Cell Signaling, cat. no. 3161), monoclonal mouse anti-RAD51 (Invitrogen, Carlsbad, CA, USA; cat. No. 35–6500), monoclonal mouse anti-cyclin B1 (BD Biosciences, cat. no. 554178) and monoclonal anti-p73 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The RNA was isolated using the RNeasy kit (Qiagen, Hilden, Germany).
6
Thymocyte Apoptosis Assay Protocol
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Thymocyte populations isolated by flow cytometry were cultured at 0.2–0.5 × 106 cells/ml in high-glucose Dulbecco's Modified Eagle's medium supplemented with 10% fetal calf serum (Bovogen, Melbourne, VIC, Australia), 50 μM 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA) and 100 μM asparagine (Sigma-Aldrich) without additional cytokines in the presence or absence of 10 μg/ml etoposide (Pfizer, Sydney, NSW, Australia), 10 μM dexamethasone phosphate (Hospira, Lake Forest, IL, USA), 10 ng/ml PMA (Sigma-Aldrich), 10 μg/ml ionomycin (Sigma-Aldrich) or following treatment with 10 Gy γ-irradiation. Cell viability was determined by flow cytometry after staining with fluorescein isothiocyanate-conjugated Annexin V and propidium iodide. Specific viability was calculated at each time point as (viability of treated cells/viability of untreated cells) × 100%. Alternatively, apoptotic cells were identified by active caspase-3 staining. Cells were fixed and permeabilised using the BD Cytofix/Cytoperm Kit for 20 min, then washed with BD Perm/Wash buffer and stained with phycoerythrin rabbit anti-active caspase-3 antibody (clone C92-605, BD Biosciences), then washed again in BD Perm/Wash buffer before analysing by flow cytometry.
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