The Smad4 silencing plasmid (siSmad4) (Forward: 5′-CGAAUACACCAACAAGUAATT-3′, Reverse: 5′-UUACUUGUUGGUGUAUAUUCGTA-3′), Smad4 overexpression plasmid and empty control plasmid (negative control, NC) were purchased from Invitrogen; Thermo Fisher Scientific, Inc. SW480 cells were seeded in a 6-well plate (1.0×105 cells /well) for 24 h before transfection and divided into three groups: i) the control (0.1% PBS) subgroup, siNC subgroup, siSmad4 subgroup, NC subgroup and Smad4 overexpression plasmid subgroup; ii) the control (0.1% PBS) subgroup, cetuximab (20 µg/ml; Merck KGaA) subgroup, NC + cetuximab (20 µg/ml) subgroup, siSmad4 + cetuximab (20 µg/ml) subgroup and siSmad4 subgroup; iii) the control (0.1% PBS) subgroup, NC + cetuximab (20 µg/ml) subgroup and Smad4 overexpression plasmid + cetuximab (20 µg/ml) subgroup. Transient transfection was effected by Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. A total of 20 µM NC, siSmad4 or Smad4 overexpression plasmid, and 5 µl Lipofectamine® 2000 were added to Opti-MEM and incubated at 25°C for 10 min. Lipofectamine® 2000 was then mixed into each well and the cells were cultured in Opti-MEM. After 6 h of culturing, the medium was replaced with RPMI-1640 containing 10% FBS. After 24 h, the cells were used to perform the subsequent experiments.
Cetuximab
Manufactured by Merck Group
Sourced in Germany, United States, China, Japan, Italy, France, United Kingdom, Netherlands, Spain, Sweden
Cetuximab is a monoclonal antibody used in laboratory settings. It functions as a targeted therapy that binds to the epidermal growth factor receptor (EGFR) on the surface of certain cells.
Automatically generated - may contain errors
Lab products found in correlation
Trastuzumab, by Roche (13 mentions)
Rituximab, by Roche (11 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Streptomycin, by Thermo Fisher Scientific (7 mentions)
Panitumumab, by Amgen (7 mentions)
Cisplatin, by Merck Group (6 mentions)
Erbitux, by Merck Group (6 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
Hct116, by American Type Culture Collection (5 mentions)
Erlotinib, by Selleck Chemicals (5 mentions)
Erlotinib, by LC Laboratories (5 mentions)
Penicillin, by Thermo Fisher Scientific (4 mentions)
Sw480, by American Type Culture Collection (4 mentions)
Epidermal growth factor (egf), by Merck Group (4 mentions)
Tocilizumab, by Roche (4 mentions)
12 well tissue culture plate, by BD (4 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (4 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Facscalibur, by BD (3 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (3 mentions)
247 protocols using cetuximab
1
Smad4 Silencing and Overexpression in Colon Cancer
2
Cetuximab Conjugation with IRDye800CW
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Cetuximab (Erbitux, Ely Lily; 2 mgml-1, 145781.6 g/mol as per FASTA sequence analysis) was conjugated to IRDye800CW (IRDye800CW-NHS Ester; Li-COR, 1166.20 g/mol) by amide coupling. A stock solution of IRDye800CW-NHS Ester (10 mg·ml-1) was prepared in anhydrous Dimethyl Sulfoxide (DMSO; Sigma-Aldrich) and added to Cetuximab (2 mg·ml-1 in phosphate buffer) at a quantity corresponding to a 10-fold molar excess of IRDye800CW to each molecule of Cetuximab. The mixture was thoroughly mixed using orbital rotation for 24h at room temperature. After conjugation, the Cet-IRDye800 was purified by removing unconjugated dye using PD-10 Desalting Columns (Cytivia) pre-equilibrated with sterile 1X DPBS (Corning). Purified Cet-IRDye800 was collected and concentrated in a 30kDa ultrafiltration tube (EMD Millipore) by centrifugation at 2,500 xg for 20 min at 4°C. The conjugated Cet-IRDye800CW was then stored at 4°C in the dark.
3
Cetuximab Response in SCCHN Tumor Models
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Cetuximab sensitive (Cetux-S) and resistant (Cetux-R) SCCHN bearing mice (tumor size: +/-200 mm³) were treated intraperitoneally with Cetuximab (Merck Serono, Darmstadt, Germany) at a dose of 30 mg/kg. Two doses of Cetuximab were given: the first was administered just after the initial 18FDG-PET and MR assessments at baseline (day 0), and the second followed one week later on day 7. Post-treatment MR and 18FDG-PET were performed on days 1 and 8. The vehicle (saline solution) was administrated in the same conditions to the control groups (CTL). Mice were distributed randomly in the different groups. For PET and MRI experiments, the investigators were blinded as to treatment group both at acquisition and at analysis. Tumor size was measured by caliper once a week and tumor volume was calculated according to the following equation:
4
Skin Prick Test for α-Gal Allergy
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Skin prick tests were conducted to a panel of commercially available food allergens (beef, pork, horse, lamb, rabbit, ostrich meats and bovine serum albumin; Bial-Aristegui Laboratory, Bilbao, Spain). cetuximab, a monoclonal antibody presenting the α-Gal oligosaccharide in the heavy chain and used for the treatment of metastatic colorectal cancer [18 (link), 39 ], was used in skin tests for diagnosis of α-Gal-induced anaphylaxis [40 (link)]. The prick test to cetuximab (Erbitux; Merck SL, Madrid, Spain) was conducted at a concentration of 5 mg/ml and intradermal tests to cetuximab were conducted with 50 μl of 1:10 (0.5 mg/ml), 1:100 (0.05 mg/ml) and 1:1000 (0.005 mg/ml) dilutions as described previously [40 (link)]. Histamine prick was used as positive skin test control.
5
Cetuximab and Radiation Exposure
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Cells were cultivated in 100 nM cetuximab- (Merck, Darmstadt) containing cell culture medium for four hours prior to radiation and permanently in cetuximab-containing cell culture medium after radiation.
Standardized radiation doses were applied using the Gulmay X-ray therapy unit D3225 using a radiation stay time table (radiation stay time based on farmer chamber measurement 30013-0415 on 31.05.2012/27.06.2012). The desired dosage (Gy) was achieved by defining radiation amount per time unit according to radiation stay time table.
Standardized radiation doses were applied using the Gulmay X-ray therapy unit D3225 using a radiation stay time table (radiation stay time based on farmer chamber measurement 30013-0415 on 31.05.2012/27.06.2012). The desired dosage (Gy) was achieved by defining radiation amount per time unit according to radiation stay time table.
6
Cetuximab and Hyperthermia Combination Therapy
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When the mouse leg tumor volume reached 70 mm3, 0.1 mg/kg cetuximab (Merck, Darmstadt, Germany) in 100 μL normal saline was systemically administered through the caudal vein.15 , 17 , 18 We used normal saline as a solution buffer for cetuximab. The tumors were then warmed in a water bath for 30 min at different temperatures: 25°C, control temperature; 37°C, intra‐abdominal organ level; and 41°C, mild hyperthermia (n = 4, each treatment group).6 , 19 The cetuximab plus heat treatment was given once every 3 days for a total of 10 applications (Fig. 2 ).18 The control group received a normal saline injection plus heat (n = 4). The size of each xenograft was determined by measuring the tumor diameter and calculating the tumor volume (V) using the following formula: V = πab2/6 where a is the maximum diameter and b is the minimum diameter.
7
Cytotoxicity and Radiosensitivity Assays
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For cisplatin, cells were seeded and, after 48 h, treated with increasing concentrations of cisplatin (Sandoz AS, Copenhagen, Denmark) for one hour. After 5 days at 37 °C under a humidified atmosphere with 5% CO2, the cell amounts were measured by the SRB assay.
For determination of cetuximab sensitivity, cells were treated with increasing concentrations of cetuximab (Merck AB, Solna, Sweden) for 5 days, after which the cell amounts were determined by the SRB assay.
Radiosensitivity was analyzed by irradiating the cells in a Gamma Cell-3000 Elan from MDS Nordion (Ottawa, ON, Canada) equipped with a Cs-137 source. After irradiation, the cells were seeded and left to grow until the untreated controls were approximately 90% confluent, and then analyzed by the SRB assay.
For all experiments the data were fitted to a sigmoidal dose-response equation with variable slope using non-linear regression, and the half maximal inhibitory concentration (IC50), inhibitory dose (ID50) or maximum inhibition (Emax) calculated in GraphPad Prism.
For determination of cetuximab sensitivity, cells were treated with increasing concentrations of cetuximab (Merck AB, Solna, Sweden) for 5 days, after which the cell amounts were determined by the SRB assay.
Radiosensitivity was analyzed by irradiating the cells in a Gamma Cell-3000 Elan from MDS Nordion (Ottawa, ON, Canada) equipped with a Cs-137 source. After irradiation, the cells were seeded and left to grow until the untreated controls were approximately 90% confluent, and then analyzed by the SRB assay.
For all experiments the data were fitted to a sigmoidal dose-response equation with variable slope using non-linear regression, and the half maximal inhibitory concentration (IC50), inhibitory dose (ID50) or maximum inhibition (Emax) calculated in GraphPad Prism.
8
Virus Neutralization Assay with Cetuximab
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Cell monolayers were exposed to the recombinant viruses at MOI ranging from 2 to 10 PFU/cell. Infection was monitored through EGFP by fluorescence microscopy. Flow cytometry (BD Accuri C6) was used to measure EGFP or the binding of MAb 52S to gH (see Supplementary Materials ). Neutralization of infection by cetuximab (Merck) was assayed by incubating cells with 83 µg/mL cetuximab 1 h before, during (1.5 h), and after (24 h) exposure to the virus.
9
Cetuximab-Mediated EGFR Inhibition in Cells
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10
Cytotoxicity Assay for HNSCC Cells
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Cytotoxicity studies were performed using gold-coated 16-well plates (E-plate16) for the xCELLigence Real-Time Cell Analysis (RTCA) (Roche Diagnostics, 00380601050), as described previously.27 (link) Seeding density was optimised for each HNSCC cell line to ensure continuous growth until the end of the assay. Briefly, cells were harvested, counted and background impedance of the E-plate16 was measured before seeding of cells. After overnight incubation, cetuximab [2 µg/ml] or human immunoglobulin G1 kappa (IgG1κ, [2 µg/ml]) (Sigma, 5154), as an isotype control was added. Additionally, either cell-free medium or isolated NK cells were added to each well at an effector (NK cells) to target (tumour cells) (E:T) ratio of 5:1. The impedance was followed up by automated measurement every 15 min starting from cell seeding and ending 48 h after treatment with cetuximab both under normoxic and hypoxic conditions. Measurement of the impedance was expressed as Cell Index (CI). Each condition was performed in dublo.
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