For all experiments, except those shown in Supplementary Figure S6 , oxaliplatin and cisplatin (Sigma-Aldrich) were dissolved in phosphate-buffered saline (PBS) to 10 mM, aliquoted and stored at -20°C until use. For experiments shown in Supplementary Figure S6c and d , oxaliplatin and cisplatin were freshly dissolved in 0.9% NaCl and in Supplementary Figure S6e , oxaliplatin was freshly dissolved in 5% dextrose (Sigma) solution. Chemical concentrations and treatment durations are indicated for each experiment. Deubiquitinase inhibitor Pierce™ NEM (N-ethylmaleimide; Thermo Fisher) was freshly prepared before each experiment by resuspending in absolute ethanol.
Oxaliplatin
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Argentina, China, France, Macao, Australia, Austria, Belgium, Italy, Sweden
Oxaliplatin is a platinum-based chemotherapeutic agent used in the treatment of various types of cancer. It functions as a DNA crosslinking agent, disrupting cellular division and leading to cell death.
Automatically generated - may contain errors
Lab products found in correlation
Cisplatin, by Merck Group (47 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (29 mentions)
5 fluorouracil, by Merck Group (23 mentions)
Carboplatin, by Merck Group (18 mentions)
Paclitaxel, by Merck Group (15 mentions)
Irinotecan, by Merck Group (14 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (13 mentions)
Doxorubicin, by Merck Group (12 mentions)
Gemcitabine, by Merck Group (12 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (12 mentions)
Hct116, by American Type Culture Collection (10 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (9 mentions)
Etoposide, by Merck Group (9 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (9 mentions)
Sn 38, by Merck Group (8 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (7 mentions)
Sw480, by American Type Culture Collection (6 mentions)
5 fluorouracil 5 fu, by Merck Group (6 mentions)
Propidium iodide, by Merck Group (6 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (5 mentions)
297 protocols using oxaliplatin
1
Optimized Drug Dissolution Protocols
2
Establishing Oxaliplatin-Resistant Colorectal Cancer Cells
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HCT116 and HT29 were cultured in RPMI-1640 medium containing 10% fetal bovine serum (Thermo Fisher, Waltham, MA, USA). To induce oxaliplatin resistance, HCT116 and HT29 were treated with 0.1 μM oxaliplatin (Sigma-Aldrich, St. Louis, MO, USA) for 9 days, and then incubated with 0.5 μM oxaliplatin for 15 days. Followed by cultured in medium containing 1 μM oxaliplatin for 30 days, the cells were exposed to 2 μM oxaliplatin to establish oxaliplatin-resistant CRC cells (HCT116/R and HT29/R) according to previous study [15 (link)]. To induce inactivation of MAPK signaling, cells were treated with 20 nM SB 203580 (Thermo Fisher) for 24 hours.
3
Colon Cancer Spheroid Culture and Chemoresistance
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The human HT-29 colonic adenocarcinoma cell line was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and was maintained in McCoy's 5A medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) at 37°C and 5% CO2, and the medium was changed every 3 days. Cells were passaged at 80% confluence and seeded at 20% confluence to keep them at optimal proliferating conditions.
For colon sphere formation, single-cell suspensions were cultured in a DMEM/F-12 basal serum-free medium (Gibco; Thermo Fisher Scientific, Inc.), containing 2 mM L-glutamine, 1 mg/ml NaHCO3, 4 µg/ml heparin, 100 µg/ml transferrin, 25 µg/ml insulin, 30 nM sodium selenite anhydrous and 20 nM progesterone (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), and supplemented with 20 ng/ml pro-epidermal growth factor (EGF; R&D Systems China Co., Ltd., Shanghai, China) and 10 ng/ml fibroblast growth factor 2 (FGF-2; R&D Systems China Co.).
In chemoresistance experiments, colon CSCs and adherent HT-29 cells were exposed to 50 µM 5-FU (Sigma-Aldrich; Merck KGaA) or 1.25 µM oxaliplatin (Sigma-Aldrich; Merck KGaA) or FOLFOX (50 µM 5-FU plus 1.25 µM oxaliplatin) for 72 h, and then observed by inverted phase-contrast microscope.
For colon sphere formation, single-cell suspensions were cultured in a DMEM/F-12 basal serum-free medium (Gibco; Thermo Fisher Scientific, Inc.), containing 2 mM L-glutamine, 1 mg/ml NaHCO3, 4 µg/ml heparin, 100 µg/ml transferrin, 25 µg/ml insulin, 30 nM sodium selenite anhydrous and 20 nM progesterone (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), and supplemented with 20 ng/ml pro-epidermal growth factor (EGF; R&D Systems China Co., Ltd., Shanghai, China) and 10 ng/ml fibroblast growth factor 2 (FGF-2; R&D Systems China Co.).
In chemoresistance experiments, colon CSCs and adherent HT-29 cells were exposed to 50 µM 5-FU (Sigma-Aldrich; Merck KGaA) or 1.25 µM oxaliplatin (Sigma-Aldrich; Merck KGaA) or FOLFOX (50 µM 5-FU plus 1.25 µM oxaliplatin) for 72 h, and then observed by inverted phase-contrast microscope.
4
Modulating miR-96 and TPM1 in CRC cell line
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The CRC cell line SW480 was purchased from the American Type Culture Collection. Cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 35˚C in an atmosphere with 5% CO2. miR-96 inhibitor, miR-negative control (miR-NC), small interfering RNA (siRNA) targeting tropomyosin 1 (TPM1) and siRNA NC were designed and provided by Guangzhou RiboBio Co., Ltd. The cells were transfected with miR-96 inhibitor, miR-NC, miR-96 mimics, siRNA TPM1 and siRNA NC using Lipofectamine™ 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) for 48 h. A total of 20 µmol/l oxaliplatin (Sigma-Aldrich; Merck KGaA) was used to investigate oxaliplatin sensitivity. The sequences of the miRNAs and siRNAs used were: miR-96 inhibitor, 5'-GCAAAAAUGUGCUAGUGCCAAA-3'; miR-NC, 5'-CAGUACUUUUGUGUAGUACAA-3'; miR-96 mimic, 5'-UUUGGCACUAGCACAUUUUUGC-3', siRNA TPM1 5'-CCCGTAAGCTGGTCATCAT-3' and siRNA NC, 5'-CCCAACGGTTGACTGTCAT-3'.
5
Oxaliplatin and bvPLA2 co-treatment protocol
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Oxaliplatin (6 mg/kg, Sigma Chemical Co, St. Louis, MO, USA) was dissolved in 5% glucose at a concentration of 2 mg/mL depending on animal weight to ensure intraperitoneal injections of ≤0.5 mL. The vehicle control group received the same volume of 5% glucose solution through the same injection route.
The mice received an intraperitoneal (i.p.) injection of bvPLA2 (Sigma) at a concentration of 0.2 mg/kg once daily for five days before Oxaliplatin was administered. The control group received an equal volume of PBS. All mice received a single injection of Oxaliplatin (6 mg/kg) two days after the last bvPLA2 or PBS injection.
The mice received an intraperitoneal (i.p.) injection of bvPLA2 (Sigma) at a concentration of 0.2 mg/kg once daily for five days before Oxaliplatin was administered. The control group received an equal volume of PBS. All mice received a single injection of Oxaliplatin (6 mg/kg) two days after the last bvPLA2 or PBS injection.
6
Establishment of Oxaliplatin-resistant HCC Cell Lines
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MHCC97H (97H), a high-metastatic human HCC cell line, was established in Liver Cancer Institute of Zhongshan Hospital [15 (link)]. MHCC97H cells from the 12th to the 15th passage were used in our experiments. Hep3B, a low metastatic potential HCC cell line was purchased from the America Type Culture Collection(ATCC, HB 8064™). The oxaliplatin-resistant HCC cell lines MHCC97H–OXA and Hep3B–OXA selected at a 25umol/L concentration of oxaliplatin were successfully established from MHCC97H and Hep3B, by exposing cells to gradually increasing oxaliplatin (Sigma, St. Louis, MO, USA) from 2 umol/L to 25umol/L in our laboratory [14 (link)]. The IC50 value of surviving HCC cells treated with oxaliplatin was about 10-fold as high as that of their parental cells (Additional file 1 : Figure S1).MHCC97H and MHCC97H–OXA were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Life Technologies/Gibco). Hep3B and Hep3B–OXA cells were cultured in minimum essential medium (MEM) supplemented with 10% fetal bovine serum. Cells(1 × 106) were seeded into 25 cm culture flask for 72 h per passage.
7
Chemotherapy Treatment Protocols in Mice
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The oxaliplatin treated mice were given two injections of oxaliplatin (10 mg/kg, ip.) (Sigma Aldrich, St. Louis MO) dissolved in phosphate buffered saline (PBS). The injections were separated by 3 days. Vehicle control animals received two injections of an equal volume of PBS. The paclitaxel treated mice received four injections of paclitaxel (26 mg/kg, ip.) (Sigma Aldrich, St. Louis MO) spaced 3 days apart. The paclitaxel was dissolved in DMSO. Vehicle treated animals received four injections of an equal volume of DMSO.
8
Rodent Models of Neuropathic Pain
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Freund’s Complete Adjuvant (CFA) [46 (link)] 20 μl emulsified with saline or alternately saline alone as vehicle control were injected into the left hindpaw plantar surface of mice. Nerve Growth Factor (NGF): 20μl of hNGF (Invitrogen) Life technologies: Recombinant Human Protein 11050-HNAC-50) (4μg / 20ul / mouse) versus saline vehicle control was intraplantarly (ipl) injected into the left hindpaw’s plantar surface [47 (link)]. A Spared Nerve Injury (SNI) model of neuropathic pain versus sham control was performed in mice through the ligation and transection of the sural and superficial peroneal branches of the sciatic nerve, leaving the tibial nerve intact as described [48 (link), 49 (link)]. Oxaliplatin-based model of neuropathic pain was accomplished by injection of mice with Oxaliplatin (Sigma) intraperitoneal (ip) 3mg/kg in saline versus saline alone vehicle control as described [50 (link)].
9
Colorectal Cancer Sphere Assay
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A total of 1 × 102 cells/well of HT-29 or 2 × 102 cells/well of SW480 were seeded in ultra-low attachment 96 well plate (Costar). Colonospheres were cultured in serum-free sphere media (DMEM/F12 supplemented with 20 ng/ml hEGF, 10 ng/ml bFGF, 1x N-2, and 1x B-27) for 6–10 days, and the number of colonospheres with a diameter of >40 μm was counted. To induce differentiation, an aliquot of spheroid HT-29 cells was trypsinized and cultured in DMEM with 10% fetal bovine serum. For oxaliplatin sensitivity assay, FLAG or FLAG-DBC1-expressing colonospheres were cultured in the presence of 10 µM oxaliplatin (Sigma-Aldrich). For extreme LDA, HT-29 or SW480 cells were plated in ultra-low attachment 96 well plates in sphere media at a density that ranged from 80 to 10 cells/well (HT-29) or ranged from 400 to 50 cells/well (SW480) (six replicates/cell concentration). After 6–10 days sphere culture, the number of wells containing colonospheres was counted, and the frequency of sphere-forming cells was determined using the Extreme Limiting Dilution Analysis software (http://www.bioinf.wehi.edu.au/software/elda/ )43 (link).
10
Oxaliplatin-induced Neuropathic Pain Model
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Oxaliplatin was purchased from Sigma (USA) and dissolved in 5% glucose/H2O as a stock solution of 1 mg/ml as a previous study reported.14 (link) Rats were intraperitoneally administered Oxaliplatin at 4 mg/kg once per day for five consecutive days to induce mechanical allodynia. The control rats received an intraperitoneal injection of an equivalent volume of 5% glucose/H2O. Intrathecal injection of the neutralizing antibody against CXCL12 (8 µg, 10 μl; Torrey Pines Biolabs, Secaucus, NJ), neutralizing antibody against TNF-α (10 µg, 10 μl; R&D systems, Minneapolis, MN), a isotype IgG (8 µg, 10 μl; Sigma, Bellevue, WA), the IL-1 receptor antagonist (IL-1ra; 50 µg, 10 μl; R&D Systems, Minneapolis, MN), a STAT3 inhibitor S3I-201 (100 µg, 10 μl; Selleckchem, Houston, TX), scramble siRNA (1 nmol, 10 μl; Ribobio, China), or CXCL12 small interfering RNA (siRNA; 1 nmol, 10 μl; Ribobio, China) for consecutive 10 days was performed 30 min before Oxaliplatin administration. In addition, recombinant rat CXCL12 (2 µg, 10 μl; Signalway Antibody, College Park, USA) were intrathecally injected for consecutive 10 days.
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