The human osteosarcoma cell lines SJSA, SAOS2, 143B, U2OS and HOS were cultured in high glucose Dulbecco’s Modified Eagle Medium (DMEM; Gibco, United States) supplemented with 10% fetal bovine serum (FBS; Wisent, Canada) and 1% Penicillin-Streptomycin-Glutamine (Wisent Inc., Cañada) in a humidified atmosphere of 5% CO2 and 95% air at 37°C. Cells were dissociated by TrypLETM Express (Gibco, Thermo Fisher Scientific Inc., United States) and passaged when they are 90%–100% confluent. Cell line authentication was performed on cells that used for in vitro and in vivo studies using Short Tandem Repeat (STR) DNA profiling and all cell lines were preserved at Shanghai Bone Tumor Institute (Shanghai, China). Reagents used in the study included: Ro-3306 (#HY-12529, MCE/MedChemExpress, United States), Doxorubicin hydrochloride (#HY-15142, MCE/MedChemExpress, United States). The antibodies used in this study were purchased as follows: anti-MYC (#ab78318, 1:1000), anti-GAPDH (#ab263962, 1:1000). HRP-conjugated goat anti-rabbit IgG (#L3042, 1:5000) and goat anti-mouse IgG (#101, 1:5000) secondary antibodies for Western blot were purchased from Signalway Antibody.
Penicillin streptomycin glutamine (psg)
Manufactured by Wisent
Sourced in Canada
Penicillin-Streptomycin-Glutamine is a sterile solution designed for use in cell and tissue culture applications. It is a combination of the antibiotics penicillin and streptomycin, along with the amino acid glutamine. This product is commonly used to prevent bacterial contamination in cell culture media.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Wisent (4 mentions)
Tryple express, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Wisent (2 mentions)
Hepes, by Wisent (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Ro 3306, by MedChemExpress (1 mentions)
Doxorubicin hydrochloride, by MedChemExpress (1 mentions)
Sr 48692, by R&D Systems (1 mentions)
Dmem f12, by Wisent (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Lance ultra camp assay kit, by PerkinElmer (1 mentions)
4 protocols using penicillin streptomycin glutamine (psg)
1
In vitro Culture of Human Osteosarcoma Cell Lines
2
Osteosarcoma Cell Lines Characterization
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Human osteosarcoma cells used in our study were from ATCC, including 143B and HOS/MNNG. In a previous study, HOS has been demonstrated as MTAP-deleted osteosarcoma and its homologous derivatives, 143B and HOS/MNNG, inherit this feature as well (García-Castellano et al., 2002 (link)). All cell lines were stored at Shanghai Bone Tumor Institution (Shanghai, China). Osteosarcoma cells were maintained in DMEM (Wisent Inc., Canada) with 10% FBS (Wisent Inc., Canada) and 1% Penicillin-Streptomycin-Glutamine (Wisent Inc., Canada) in a humidified atmosphere of 5% CO2 and 95% air at 37°C. Cells were dissociated by TrypLETM Express (Gibco, Thermo Fisher Scientific Inc., United States) and passaged when they are 90–100% confluent. All cell lines were certificated by short tandem repeat (STR) analysis.
3
Synthesis and Characterization of NTS1 Antagonist Pepducins
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In order to perform the experiments described here, we obtained the NTS1 antagonist SR48692 from R & D Systems, Inc. (Minneapolis, MN, USA). We synthesized the compounds NT(8-13), and our NTS1-derived pepducin series at the peptide synthesis facility of the Université de Sherbrooke (http://www.usherbrooke.ca/ips/en/platforms/psp/). This series consists of five main pepducins (NTS1-ICL1-PP-001 to NTS1-ICL1-PP-005), a non-palmitoylated control (NTS1-ICL1-NP-001), a pepducin with a scrambled peptide sequence (NTS1-ICL1-PP-SCR-001), as well as a TAMRA-labeled pepducin (NTS1-ICL1-FP-001). The specific peptide sequences are provided in Table 1. The protected amino acids and TentaGel R-RAM resins used in their synthesis were obtained from ChemImpex International (Wood Dale, IL, USA). All other chemicals and reagents involved in peptide and pepducin synthesis were obtained from Sigma-Aldrich (Oakville, ON, Canada) or Fisher Scientific (Montreal, QC, Canada). DMEM-F12, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), penicillin-streptomycin-glutamine, fetal bovine serum (FBS), gentamycin G418, phosphate-buffered solution (PBS), Hank's balanced salt solution (HBSS), trypsin-EDTA 0.25% were all obtained from Wisent (St. Bruno, QC, Canada).
4
Synthesis and Characterization of Apelin-13
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All compounds including apelin-13 were synthesized by us as previously described [19] (link).
Coelenterazine 400A (DeepBlueC) was purchased from Gold Biotechnology Inc. (St. Louis, MO, USA). DMEM, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), Penicillinstreptomycin-glutamine and fetal bovine serum (FBS) were obtained from Wisent (St. Bruno, QC, Canada), Opti-MEM was acquired from Invitrogen (Burlington, ON, Canada). Lance Ultra cAMP assay kit was purchased from Perkin Elmer (Montréal, QC, Canada).
Coelenterazine 400A (DeepBlueC) was purchased from Gold Biotechnology Inc. (St. Louis, MO, USA). DMEM, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), Penicillinstreptomycin-glutamine and fetal bovine serum (FBS) were obtained from Wisent (St. Bruno, QC, Canada), Opti-MEM was acquired from Invitrogen (Burlington, ON, Canada). Lance Ultra cAMP assay kit was purchased from Perkin Elmer (Montréal, QC, Canada).
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