The U2OS cell line used in these studies was the human osteosarcoma cell line, obtained directly from ATCC (HTB96). Cells were authenticated by morphological assessment under microscopy. Cells were grown in a 37°C, 5% CO2 tissue culture incubator on tissue culture treated dishes in DMEM + 10% FCS and passaged with Trypsin EDTA.
Htb 96
Manufactured by American Type Culture Collection
Sourced in United States, France
The HTB-96 is a 96-well plate designed for high-throughput cell culture and experimentation. It provides a standardized platform for cultivating and analyzing cells in a multi-well format. The product's core function is to facilitate the uniform growth and maintenance of cell lines in a 96-well configuration, enabling researchers to conduct parallel experiments and assays.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (12 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (12 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (9 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (6 mentions)
Hb 8065, by American Type Culture Collection (5 mentions)
Fetal bovine serum (fbs), by Merck Group (5 mentions)
Crl 3216, by American Type Culture Collection (4 mentions)
Ccl 2, by American Type Culture Collection (4 mentions)
Ccl 185, by American Type Culture Collection (4 mentions)
Streptomycin, by Merck Group (4 mentions)
Penicillin, by Merck Group (4 mentions)
Ccl 247, by American Type Culture Collection (3 mentions)
Dmem f12, by Thermo Fisher Scientific (3 mentions)
Mccoy s 5a modified medium, by Thermo Fisher Scientific (3 mentions)
L glutamine, by Merck Group (3 mentions)
Crl 4000, by American Type Culture Collection (3 mentions)
Rpmi 1640 medium, by American Type Culture Collection (3 mentions)
Ccl 81, by American Type Culture Collection (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
96 protocols using htb 96
1
Culturing U2OS Osteosarcoma Cells
2
Propagation of GPCMV and HSV-1 Viruses
3
Cell Culture and Synchronization Protocols
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HeLa cells, HEK293 cells, and U2OS (an osteosarcoma cell line, purchased from ATCC, HTB-96) cells were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum and 5% CO2. HEK293 cells stably expressing Cdt2-FLAG were described previously [30 (link)]. HEK293 cells stably expressing Cdt1-3NLSmyc or PIP-box mutated Cdt1 (A6-Cdt2-3NLSmyc) were isolated after transfection with pCMV-Cdt1-3NLSmyc or pCMV-A6-Cdt1-3NLSmyc [8 (link)], respectively. HeLa cells were synchronized in M phase after double thymidine block (first thymidine 2 mM for 15 h, release for 9 h, and second thymidine for 15 h and released in the presence of nocodazole (40 ng/ml) for 9 h; U2OS cells after single thymidine block (2 mM thymidine for 15 h), and released in the presence of nocodazole (40 ng/ml) for 12 h; HEK293 cells after double thymidine block (first thymidine 2 mM for 15 h, release for 8 h, and second thymidine for 15 h) and released in the presence of nocodazole (400 ng/ml) for 12 h). UV-C (254 nm) irradiation of whole cells in dishes was performed in the presence of 5 ml Dulbecco’s modified Eagle’s medium in 10-cm dishes at 20 to 100 J/m2 using a UV cross-linker (FS-800, Funakoshi). Methyl methane sulfonate (MMS) was used at 1mM. Flow cytometry analysis of cell cycle was performed as described previously [30 (link)].
4
Authentication and Culture of U2OS Cells
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U2OS cells were purchased from ATCC (HTB-96) and periodically authenticated by short tandem repeat (STR) profiling. U2OS cells were cultured in Dulbecco’s modified Eagle’s medium (HyClone) supplemented with 10% fetal bovine serum (HyClone SH30071.03 and SH30396.03), 1X GlutaMAX (Thermo Fisher Scientific 35050061), 50 U/ml penicillin, and 50 μg/ml streptomycin (Gibco 15140–122), and maintained at 37°C in a humidified incubator with 5% CO2. FuGENE 6 (Promega E2691) was used for transient transfections per the manufacturer’s instructions. G3BP1/2 KO cells have been previously described (Zhang et al., 2018 (link)). U2OS cells stably expressing G3BP1-GFP have been previously described (Figley et al., 2014 (link)). Cells were checked for mycoplasma with MycoAlert Mycoplasma Detection Kit (Lonza LT07-318) and then regularly checked for mycoplasma by DAPI staining.
5
Live-Virus Neutralization Assay for COVID-19 Sera
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A neutralization assay was performed on sera collected at M11-13 from a panel of 28 COVID-19 positive HCW, including 13 who had received a single dose of COVID-19 vaccine. Live-virus neutralization was analyzed using the S-Fuse reporter cells, as previously reported [20] (link). Briefly, S-Fuse reporter cells correspond to U2OS cells (ATCC Cat# HTB-96, RRID: CVCL_0042) engineered to express ACE2 and either GFP1–10 or GFP11. When mixed, these cells produce GFP upon syncytia formation which occurs during productive infection with SARS-CoV-2. Neutralization of infectious D614G, B.1.1.7, and B.1.351 variants grown in Vero E6 cell lines (ATCC Cat# CRL-1586, RRID: CVCL_0574) was assessed for each serum using limiting dilutions. Infection was quantified by measuring the number of GFP+ syncytia 18 h after infection. The percentage of neutralization was calculated using the number of syncytia as the value with the following formula: . Neutralizing activity of each serum was expressed as the half-maximal inhibitory concentration (IC50).
6
Cultivation of U2OS and NIH3T3 Cell Lines
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U2OS cells (human osteosarcoma, ATCC #HTB-96), U2OSR1 and U2OSR1 K514R cells (U2OS cells stably transfected with FGFR1 or FGFR1 K514R, kind gift of Dr. E.M. Haugsten from the Department of Molecular Cell Biology (Institute for Cancer Research, Oslo University Hospital)), were cultivated in Dulbecco’s Modified Eagle’s Medium (Biowest, Nuaille, France) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA), antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin). For U2OSR1 cells growth media were additionally supplemented with geneticin (1 mg/ml). NIH3T3 (murine embryonic fibroblasts, ATCC #CRL-1658) were grown in Dulbecco’s Modified Eagle’s Medium (Biowest, Nuaille, France) supplemented with 2% bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) and antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin). Cells were cultivated in 5% CO2 atmosphere at 37 °C. Cells were seeded into tissue culture plates one day prior start of the experiments.
7
Generation of PonA-inducible Cell Lines
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Human U2OS PonA or Flp-In U2OS PonA cell line was generated from U2OS cells (ATCC, HTB-96™) or Flp-In U2OS cells (gift from Dr. Senecal34 (link)) by stable transfection of pERV3, which expresses synthetic VP16-glucocorticoid/ecdysone receptor (VgEcR) and retinoid-X-receptor (RXR) that are required for induction for the transcription of PonA promoter as described previous study73 (link). Human embryonic kidney 293 T PonA cell was generated from HEK 293 T cell (ATCC, CRL-3216™) by stable transfection of pERV3-zeocin vector, which is replaced neomycin drug selection gene in pERV3 to Zeocin gene due to the neomycin resistance of HEK293T cells. Both cell lines were grown at 37 °C and 5% CO2 in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 4.5 g/l of glucose, 10% FBS and 1% penicillin-streptomycin.
8
Induction and Quantification of Autophagy in Cancer Cell Lines
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Culture media and supplements for cell culture were purchased from Gibco-Invitrogen (Carlsbad, CA, USA) and plasticware from Corning (Corning, NY, USA). Human osteosarcoma U2OS cells (cell line was purchased from ATCC [HTB-96]) and their GFP-LC3-expressing derivatives were cultured in DMEM medium containing 10% foetal bovine serum, 100 mg/l sodium pyruvate, 10 mM HEPES buffer, 100 units/ml penicillin G sodium and 100 μg/ml streptomycin sulphate (37 °C, 5% CO2). Human hepatocellular carcinoma HepG2 cells (cell line was purchased from ATCC [HB8065]) were cultured in EMEM medium supplemented with 10% fetal bovine serum, 100 mg/l sodium pyruvate, 10 mM HEPES buffer, 100 units/ml penicillin G sodium and 100 μg/ml streptomycin sulphate. Human colorectal cancer HCT116 cells (cell line was purchased from ATCC [CCL-247]) were cultured in McCoy’s medium enriched with 10% fetal bovine serum, 100 mg/l sodium pyruvate, 10 mM HEPES buffer, 100 units/ml penicillin G sodium and 100 μg/ml streptomycin sulfate. For autophagy induction, cells were treated for 8 h with 50 μM DMC (Sigma Aldrich [S617237]) in presence or absence of 50 μM chloroquine (Sigma Aldrich [C6628]) to properly assess autophagic flux.
9
Culturing Human Osteoblast and Cancer Cell Lines
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The human normal osteoblast cell line hFOB 1.19 (ATCC® CRL-11372™), the OS cell lines U2OS (ATCC® HTB-96™), MG-63 (ATCC® CRL-1427™) and Saos-2 cells (ATCC® HTB-85™), and normal human 293 cells (ATCC® CRL-1573™) were obtained from ATCC. All cells were maintained in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% heat-inactivated FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin solution (Sigma-Aldrich; Merck KGaA) in a humidified incubator with 5% CO2 at 37°C.
10
Comparative Cell Culture Protocol for Hepatic and Non-Hepatic Cell Lines
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HEK293 (ATCC® CRL-1573™), A549 (ATCC® CCL-185™), HeLa (ATCC® CCL-2™), U2OS (ATCC® HTB-96), HepG2 (ATCC® HB-8065) and HepG2-NTCP-K7 [10] cell lines were maintained in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12, Gibco, Carlsbad, USA) supplemented with 10% heat-inactivated fetal calf serum (FCS; Gibco) and 1% penicillin/streptomycin (10.000 U/mL, Gibco) in a humidified 37 °C, 5% CO2 incubator. HepaRG cell line (RRID:CVCL_9720) was cultivated in William’s E medium supplemented with 10% FBS Fetalclone II (Thermo Fisher Scientific, Waltham, MA, USA), 1% penicillin/streptomycin, 2 mM glutamine, 0.023 U/mL human insulin, 0.0047 mg/mL hydrocortisone and 0.08/mL gentamicin (Gibco). HepG2, HepG2-NTCP-K7 and HepaRG cells were cultivated on collagen-coated plates, HEK293 cell line on poly-l -lysine coated plates. To differentiate HepG2 and HepG2-NTCP-K7 cells, 2.5% DMSO was added to the cell culture medium for 3 days when cells were sub confluent. For differentiation of HepaRG, cells were first cultivated for 2 weeks in standard medium, followed by cultivation in medium supplemented with 1.8% DMSO for another 2 weeks. All experiments were performed in 24 well plates using 500 µL of standard cultivation medium per well.
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