Human NB cell lines UKF-NB-3 and UKF-NB-4 [40 ], established from bone marrow metastases of high-risk NB, were a gift from Prof. J. Cinatl, Jr. (J. W. Goethe University, Frankfurt, Germany). IMR-32 and SH-SY5Y were purchased from ECACC, Salisbury, UK. Cells were cultivated in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (all from Gibco Life Technologies, Carlsbad, CA, USA). Cultured cells were grown in a humid incubator at 37°C and 5% CO2. Sodium salt of valproic acid (VPA), trichostatin-A (TSA), vorinostate (SAHA), entinostat (MS-275) and valpromide (VPM) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). VPA was dissolved in IMDM while other HDAC inhibitors (TSA, SAHA and MS-275) and VPA analogue (VPM) were dissolved in DMSO that was used in final concentration not exceeding 0.3%. Vincristine sulfate (VCR) and cisplatin (CDDP) were obtained from Teva Pharmaceuticals (Prague, Czech Republic) and PLIVA-Lachema (Brno, Czech Republic) respectively.
Cisplatin cddp
Manufactured by Teva Pharmaceuticals
Sourced in Germany, Israel
Cisplatin (CDDP) is a platinum-based chemotherapeutic agent used in the treatment of various types of cancer. It functions as a DNA-binding agent, interfering with cell division and leading to cell death.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Iscove s modified dulbecco s medium imdm, by Thermo Fisher Scientific (1 mentions)
X rad 320 ix, by Precision X-Ray (1 mentions)
Anti p21, by Merck Group (1 mentions)
Doxorubicin, by Pfizer (1 mentions)
Anti p53 do 1, by Santa Cruz Biotechnology (1 mentions)
P histone h2a x s139, by Cell Signaling Technology (1 mentions)
N acetylcysteine nac, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Anti phospho p53, by Cell Signaling Technology (1 mentions)
Anti parp, by Cell Signaling Technology (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Doxorubicin, by Teva Pharmaceuticals (1 mentions)
4 protocols using cisplatin cddp
1
Neuroblastoma Cell Line Culture Protocol
2
Optimized Treatment Conditions for Bortezomib and Cisplatin
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Bortezomib (BZM; Cell Signaling Technology, Danvers, MA) was diluted in dimethyl sulfoxide (DMSO, stock: 1 mM) according to the manufacturer's instructions and stored at −20°C upon use. Further dilution steps were carried out directly before application, and an equal dilution of DMSO was used as solvent control. Cisplatin (CDDP; TEVA, Ulm, Germany) was supplied as a stock solution (1 mg/ml) (Center for Cytostatics Preparation, University Hospital Gießen and Marburg, Germany) and further diluted in pure water (stock: 1 mM) directly before application. X-ray irradiation (IR) was carried out using an X-RAD 320 iX (Precision X-Ray Inc., Denver, CO) X-ray tube; anode voltage: 320 kV, current: 10 mA, dose rate: 1.2 Gy/min, focus object distance: 60 cm, filter: 0.5 mm Cu and 0.5 mm Al. Further treatment conditions are indicated in the results section.
3
Evaluation of NAC-mediated Chemosensitization
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N-acetyl cysteine (NAC) were purchased from Sigma-Aldrich (St. Louis, MO). Carboplatin (CBP) (SAGENT Pharmaceuticals, Schaumburg, IL), cisplatin (CDDP) (Teva Pharmaceuticals, Sellersville, PA) and doxorubicin (Pfizer Inc, New York, NY) were obtained from the institutional inpatient pharmacy. The following antibodies were used in this study: anti-p53 DO-1(Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho- P53 (Cell Signaling, Danvers, MA, USA), anti-p21 (EMD Millipore, Billerica, MA, USA), anti-PARP (Cell Signaling, Danvers, MA, USA), ATM (Santa Cruz Biotechnology, Santa Cruz, CA, USA), p-ATM (EMD Millipore, Billerica, MA, USA), p-Histone H2AX S139 (Cell signaling, Danvers, MA, USA), LDHa, LDHb (Abcam, Cambridge, MA, USA) and β-actin (Sigma, St. Louis, MO, USA).
4
Cytotoxicity Evaluation of CDDP and Doxorubicin
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Cisplatin (CDDP) and doxorubicin were purchased from Teva (Israel) and diluted in a complete culture medium. For chemosensitivity analysis, Neuro2a cells were seeded at 70% confluence and treated with 30 μM CDDP. The CDDP working concentration was determined through comparing the cell apoptosis level in treated cells being significantly higher with that in non-treated control WT cells. The cells treated with PBS were used as a control for relative comparison. After 24 h of treatment, cells were collected for flow cytometry, western blot analysis and caspase activity assay.
The effect of doxorubicin on cell survival was analyzed using Neuro2a cells, which were seeded in a 96-well plate at a concentration of 1 × 104 cells/well in 100 μL of culture medium and treated with 1 μM doxorubicin. MTT test was performed to evaluate cell viability before doxorubicin administration (0 h) and 24 h after as described earlier [24 (link)]. Briefly, at regular time points the cells were incubated with 0.1 mg of MTT (Merck Millipore, Burlington, MA, USA) for 2 h at 37 °C and then lysed in 100 μL of lysing buffer (25 mM HCl, 2% acetic acid, 3% DMF, 5% SDS, pH 4.7). The absorbance at 570 nm was measured using plate reader Infinite F200 PRO (Life Sciences, Tecan, Grodig, Austria).
The effect of doxorubicin on cell survival was analyzed using Neuro2a cells, which were seeded in a 96-well plate at a concentration of 1 × 104 cells/well in 100 μL of culture medium and treated with 1 μM doxorubicin. MTT test was performed to evaluate cell viability before doxorubicin administration (0 h) and 24 h after as described earlier [24 (link)]. Briefly, at regular time points the cells were incubated with 0.1 mg of MTT (Merck Millipore, Burlington, MA, USA) for 2 h at 37 °C and then lysed in 100 μL of lysing buffer (25 mM HCl, 2% acetic acid, 3% DMF, 5% SDS, pH 4.7). The absorbance at 570 nm was measured using plate reader Infinite F200 PRO (Life Sciences, Tecan, Grodig, Austria).
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