U2OS (ATCC htb-96; ATCC, Manassas, VA) cells were maintained in Dulbecco’s modified essential medium (DMEM) supplemented with 5% (vol/vol) fetal calf serum, 5% (vol/vol) bovine calf serum, and 2 mM l -glutamine at 37°C. The HSV-1 n212 ICP0 nonsense mutant virus, which contains a nonsense mutation in codon 212, has been described previously (44 (link)). The n212R ICP0+ rescued virus was constructed by homologous recombination of a full-length ICP0-containing plasmid with infectious n212 virus (6 (link)). The dProm ICP0-promoter deletion mutant virus and the corresponding PromR ICP0+ rescued virus were constructed by homologous recombination with the ICP0-null 7134 virus (6 (link)). Stocks of ICP0− and ICP0+ viruses were propagated and titrated in U2OS cells.
U2os atcc htb 96
Manufactured by American Type Culture Collection
Sourced in United States
U2OS (ATCC HTB-96) is a well-characterized human osteosarcoma cell line. It is a commonly used model for cell biology and cancer research studies.
Automatically generated - may contain errors
Lab products found in correlation
Centrifuge 5810, by Eppendorf (2 mentions)
Ccl 131, by American Type Culture Collection (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Mg 63 atcc crl 1427, by American Type Culture Collection (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Saos 2, by Thermo Fisher Scientific (1 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dmem f12 glutamax modified medium, by Thermo Fisher Scientific (1 mentions)
Saos 2, by American Type Culture Collection (1 mentions)
Mccoy s 5a modified medium, by Thermo Fisher Scientific (1 mentions)
3t3 l1 atcc cl 173, by American Type Culture Collection (1 mentions)
Trypsin edta solution, by Thermo Fisher Scientific (1 mentions)
Mcf 7 atcc htb 22, by American Type Culture Collection (1 mentions)
Mda mb 231 atcc htb 26, by American Type Culture Collection (1 mentions)
Erlotinib sml2156, by Merck Group (1 mentions)
Sc 37007, by Santa Cruz Biotechnology (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Draq5, by Biostatus (1 mentions)
Operetta cls, by PerkinElmer (1 mentions)
9 protocols using u2os atcc htb 96
1
Characterization of HSV-1 ICP0 Mutant Viruses
2
Cultivation of Human Osteosarcoma Cell Lines
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Human osteosarcoma cells 143B (ATCC CRL-8303) and U2OS (ATCC HTB-96) were purchased from National Collection of Authenticated Cell Cultures (Shanghai, China) at July, 2020. The cell lines have been authenticated by STR (Short Tandem Repeat) authentication and tested for mycoplasma contamination by National Collection of Authenticated Cell Cultures (Shanghai, China) before the purchase. The 143B cells were cultured in RMPI-1640 medium supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin. The U2OS cells were cultured in Mccoy’s 5A medium supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin. The cells were maintained in an incubator at 37 ℃ with 5% CO2. The medium was replaced every 2 days during the culture.
3
Culturing and Harvesting U-2 OS and Neuro-2a Cells
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U2OS human osteosarcoma bone cells (U-2 OS, ATCC® HTB-96) were purchased from ATCC. Neuro-2a mouse neuroblasts cells (ATCC® CCL-131™) were also used as a control in some experiments. Cells were grown in Tissue Culture incubators (Forma Scientific CO2, water, jacketed incubator; Life Sciences Instruments), in pre-warmed (37°C) 1xDMEM (containing 4,500 mg/L glucose, 110 mg/L sodium pyruvate, 584 mg/L L-glutamine and no HEPES) (Dulbecco's Modified Eagle Medium) (Thermo Scientific/Gibco, 41966-052) medium complemented with FBS (Thermo Fisher Scientific/Gibco, 10270-106) (10% v/v/), and 100 U/ml of penicillin and streptomycin (P/S) ((Fisher Scientific/Gibco, 15140-122) (1% v/v/), at 37°C, 5% CO
2. Cells washes were carried out with 1xDPBS (Thermo Scientific/Gibco, 14190-169) pre-warmed at 37°C.
Cells were harvested, before reaching confluency, after Trypsin-EDTA (0.05%) (Gibco, 25300-054) digest at 37°C, for 5 min; digest was stopped by the addition of 1xDMEM (pre-warmed at 37°C). Cells were spun in a centrifuge (Eppendorf centrifuge 5810) at 211xG (1.000 rpm), for 5 min at room temperature (RT), and the supernatant removed. The cell pellet was either immediately frozen in liquid nitrogen or was resuspended in a small volume of 1xDMEM and fixed in ice cold (-20°C) 70% ethanol solution overnight (or for several days) at -20°C.
2. Cells washes were carried out with 1xDPBS (Thermo Scientific/Gibco, 14190-169) pre-warmed at 37°C.
Cells were harvested, before reaching confluency, after Trypsin-EDTA (0.05%) (Gibco, 25300-054) digest at 37°C, for 5 min; digest was stopped by the addition of 1xDMEM (pre-warmed at 37°C). Cells were spun in a centrifuge (Eppendorf centrifuge 5810) at 211xG (1.000 rpm), for 5 min at room temperature (RT), and the supernatant removed. The cell pellet was either immediately frozen in liquid nitrogen or was resuspended in a small volume of 1xDMEM and fixed in ice cold (-20°C) 70% ethanol solution overnight (or for several days) at -20°C.
4
Culturing and Harvesting U-2 OS and Neuro-2a Cells
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U2OS human osteosarcoma bone cells (U-2 OS, ATCC® HTB-96) were purchased from ATCC. Neuro-2a mouse neuroblasts cells (ATCC® CCL-131™) were also used as a control in some experiments. Cells were grown in Tissue Culture incubators (Forma Scientific CO2, water, jacketed incubator; Life Sciences Instruments), in pre-warmed (37°C) 1xDMEM (containing 4,500 mg/L glucose, 110 mg/L sodium pyruvate, 584 mg/L L-glutamine and no HEPES) (Dulbecco's Modified Eagle Medium) (Thermo Scientific/Gibco, 41966-052) medium complemented with FBS (Thermo Fisher Scientific/Gibco, 10270-106) (10% v/v/), and 100 U/ml of penicillin and streptomycin (P/S) ((Fisher Scientific/Gibco, 15140-122) (1% v/v/), at 37°C, 5% CO
2. Cells washes were carried out with 1xDPBS (Thermo Scientific/Gibco, 14190-169) pre-warmed at 37°C.
Cells were harvested, before reaching confluency, after Trypsin-EDTA (0.05%) (Gibco, 25300-054) digest at 37°C, for 5 min; digest was stopped by the addition of 1xDMEM (pre-warmed at 37°C). Cells were spun in a centrifuge (Eppendorf centrifuge 5810) at 211xG (1.000 rpm), for 5 min at room temperature (RT), and the supernatant removed. The cell pellet was either immediately frozen in liquid nitrogen or was resuspended in a small volume of 1xDMEM and fixed in ice cold (-20°C) 70% ethanol solution overnight (or for several days) at -20°C.
2. Cells washes were carried out with 1xDPBS (Thermo Scientific/Gibco, 14190-169) pre-warmed at 37°C.
Cells were harvested, before reaching confluency, after Trypsin-EDTA (0.05%) (Gibco, 25300-054) digest at 37°C, for 5 min; digest was stopped by the addition of 1xDMEM (pre-warmed at 37°C). Cells were spun in a centrifuge (Eppendorf centrifuge 5810) at 211xG (1.000 rpm), for 5 min at room temperature (RT), and the supernatant removed. The cell pellet was either immediately frozen in liquid nitrogen or was resuspended in a small volume of 1xDMEM and fixed in ice cold (-20°C) 70% ethanol solution overnight (or for several days) at -20°C.
5
Culturing Human Osteosarcoma Cell Lines
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Human Osteosarcoma cell lines MG63 (ATCC® CRL1427™), U-2OS (ATCC® HTB-96™), and SAOS-2 (ATCC® HTB-85™), purchased from American Type Culture Collection (ATCC), were used. MG63 cell line was cultured in DMEM F12-GlutaMAX™ Modified Medium (Gibco) supplemented with 10% Foetal Bovine Serum (FBS) (Gibco) and 1% of penicillin/streptomycin mixture (pen/strep) (100 U/ml—100 μg/ml, Gibco). SAOS-2 and U-2OS cell lines were cultured in McCoy’s 5A Modified Medium (Gibco) supplemented with 15 and 10% FBS, respectively, and 1% pen/strep. Cells were kept in an incubator at 37°C under controlled humidity and 5% CO2 atmosphere conditions. Cells were detached from culture flasks by trypsinization and centrifuged. The cell number and viability were determined by Trypan Blue Dye Exclusion test and all cell handling procedures were performed under a laminar flow hood in sterility conditions. For the experiment, all cell lines were seeded 5.0 × 103 cells/well in 96 well-plates and 5.0 × 104 cells/well in 6 well-plates.
6
Cell Culture Conditions for 3T3 L1 and U2OS
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3T3 L1 (ATCC CL-173) and U2OS (ATCC HTB-96) cells were obtained from the commercial vendor American Type Culture Collection. The U2OS-Bmal1-luc cell line has been described previously (27 (link)). All cells were maintained at 37 °C and 5% CO2 in DMEM supplemented with 10% FBS.
7
Osteosarcoma and Breast/Lung Cancer Cell Lines
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MG-63 (ATCC® CRL-1427™) and U-2OS (ATCC® HTB-96™) human osteosarcoma cell lines were obtained from the American Type Culture Collection. Human breast MCF-7 (ATCC® HTB-22™) and MDA-MB-231 (ATCC® HTB-26™) or lung H-125 (ATCC® CRL-5801™) cancer cell lines were used as controls for in vitro or in vivo AVPR2 expression and were also acquired from the American Type Culture Collection. Tumor cell lines were grown in Dulbecco's modified Eagle's medium (DMEM, Thermo Fisher Scientific Inc.) plus 10% fetal bovine serum (FBS, Natocor), 2 mM glutamine and 80 µg/ml gentamycin in monolayer culture, at 37˚C in a humidified atmosphere of 5% CO2. Cells were harvested using a trypsin/EDTA solution (Thermo Fisher Scientific Inc.) diluted in PBS and routinely tested for mycoplasma.
8
Osteosarcoma Cell Line Experiments
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The human osteosarcoma cell lines MG63 (ATCC® CRL-1427™) and U2OS (ATCC® HTB-96™) were obtained from American Type Culture Collection (Manassas, VA, USA), and maintained in Dulbecco’s Modified Eagle Medium (DMEM, Sigma–Aldrich, St. Louis, MO, USA). All media were supplemented with 10% fetal bovine serum (FBS, Sigma–Aldrich). These cell lines were employed for the described experiments without further authentication. Sodium cantharidate (CN100506212C) was obtained from Shandong Luoxin Pharmaceutical Co., Ltd (Linyi, Shandong, China). LY5 (#562712) was purchased from MedKoo Biosciences, Inc. (Morrisville, NC, USA). Erlotinib (SML2156) was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd (Shanghai, China). The lentivirus expressing a constitutively active mutant form of STAT3 (EF.STAT3C.Ubc.GFP) was a gift from Linzhao Cheng (Addgene plasmid #24983; http://n2t.net/addgene:24983 ; RRID: Addgene_24983) [53 (link)]. Vector pLenti-CMV-MCS-GFP-SV-puro was a gift from Paul Odgren (Addgene plasmid #73582; http://n2t.net/addgene:73582 ; RRID: Addgene_73582) [54 (link)]. Negative control siRNAs (sc-37007) and STAT-3-targeted siRNA (siSTAT3; sc-29493) were obtained from Santa Cruz Biotechnology, Inc. (Shanghai, China).
9
Quantitative Imaging of Osteosarcoma and Trypanosoma Infection
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Human osteosarcoma cell line U2OS (ATCC HTB-96) and rhesus monkey kidney epithelial cell LLM-MK2 (ATCC CCL-7) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). T. cruzi Y strain was maintained as tissue culture trypomastigotes (TCT) by infection to LLC-MK2 cell line. The U2OS human cell was cultivated in DMEM-high-glucose media supplemented with 10% heat-inactivated fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA, USA). To visualize amastigote replication, U2OS cells were seeded in a 384-well plate (4 × 103 cells/well) and infected with 5 × 104 TCT parasites in DMEM-low-glucose media supplemented with 2% heat-inactivated FBS as previously described [37 (link)]. The reference drug 400 μM of benznidazole (positive control), 0.5% DMSO (negative control), and the tested samples were treated on the day of assay and then incubated for 3 days at 37 °C, 5% CO2. After incubation was completed, the cells and parasites were fixed with 4% paraformaldehyde (PFA), and the DNA was stained with 5 μM DRAQ5 (Biostatus, Shepshed, Leicestershire, UK) followed by image acquisition using an automated confocal microscope (Operetta CLS, PerkinElmer, Waltham, MA, USA) with a 635 mm laser at 20× objective.
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