U2os htb 96

Manufactured by American Type Culture Collection

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U2OS (HTB-96) is a human bone osteosarcoma cell line. It is an adherent cell line derived from the primary tumor of a 15-year-old female Caucasian.

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Lab products found in correlation

Fetal bovine serum (fbs), by Thermo Fisher Scientific (9 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (5 mentions) Pen strep, by Thermo Fisher Scientific (5 mentions) Saos 2 htb 85, by American Type Culture Collection (4 mentions) Hek293t, by American Type Culture Collection (4 mentions) Penicillin, by Thermo Fisher Scientific (3 mentions) Streptomycin, by Thermo Fisher Scientific (3 mentions) Mccoy s 5a medium, by American Type Culture Collection (2 mentions) Anti ikkα, by Cell Signaling Technology (2 mentions) Anti lexa, by Merck Group (2 mentions) Anti hoip, by R&D Systems (2 mentions) Anti rbck1 hoil 1l, by Merck Group (2 mentions) Anti tradd 3684, by Cell Signaling Technology (2 mentions) Anti ikbkg nemo antibodies, by Merck Group (2 mentions) Anti actin, by Merck Group (2 mentions) Anti traf2, by Cell Signaling Technology (2 mentions) Anti tnfr1, by Cell Signaling Technology (2 mentions) Anti tak1 4505, by Cell Signaling Technology (2 mentions) Anti gfp, by Takara Bio (2 mentions) Anti flag m2, by Merck Group (2 mentions)

Close spelling variants of this product

HTB-96 (96 citations) U2OS (ATCC HTB-96) (9 citations) HTB-96TM (8 citations) U2OS osteosarcoma cells (HTB-96 (2 citations)

26 protocols using u2os htb 96

Cultivation of Human Osteosarcoma Cell Lines

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Three human osteosarcoma cell lines, Saos-2 (HTB-85), MG-63 (CRL-1427), and U2OS (HTB-96) were procured from ATCC (Manassas, USA). Saos-2 and U2OS cells were cultivated in McCoy’s 5A Medium (30–2007, ATCC) and added with 10% FBS (Gibco, Waltham, USA). MG-63 cells were cultivated in Eagle’s Minimum Essential Medium (EMEM, 30–2003, ATCC) and added with 10% FBS (Gibco). Normal human osteoblast hFOB1.19 (CRL-11372) was procured from ATCC and cultivated in a 1:1 mixture of Ham’s F12 Medium Dulbecco’s Modified Eagle’s Medium (ATCC) containing 10% FBS (Gibco). All cell types were cultivated at 37°C in a humidified atmosphere containing 5% CO₂.

Zhang Q., Tang X., Zhou Y., Chen X., Peng K., Jiang R., Liu Z., Song X, & Xia H. (2023). LINC01060 knockdown inhibits osteosarcoma cell malignant behaviors in vitro and tumor growth and metastasis in vivo through the PI3K/Akt signaling. Cancer Biology & Therapy, 24(1), 2198904.

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Mycoplasma-free Cell Line Authentication

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U2OS (HTB-96) and HEK293T (CRL-11268) were purchased from ATCC and HeLa were authenticated using Short Tandem Repeat (STR) analysis by ATCC services (100% match). All the cells lines used were uninfected with Mycoplasma, as routinely verified using the e-Myco Mycoplasma kit from FroggaBio.

Deveryshetty J., Peterlini T., Ryzhikov M., Brahiti N., Dellaire G., Masson J.Y, & Korolev S. (2019). Novel RNA and DNA strand exchange activity of the PALB2 DNA binding domain and its critical role for DNA repair in cells. eLife, 8, e44063.

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Comprehensive Antibody Validation Protocol

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Antibodies used are anti-LexA (06-719, Millipore), anti-Myc 9E10 (MMS-150P, Covance), anti-Flag M2 (A8592, Sigma Aldrich), anti-GFP (632592, Clontech), anti-Actin (A2066, Sigma Aldrich), anti-HOIP (MAB8039, R&D systems), anti-RBCK1/HOIL-1L (HPA024185, Sigma Aldrich). Anti-Phospho-IκBα (2859), anti-IκBα (4814), anti-Phospho-p65 (3033), anti-p65 (8242), anti-TRADD (3684), anti-TRAF2 (4712), anti-IKKα (11930), anti-TNF-R1 (3736) and anti-TAK1 (4505) were purchased from Cell Signaling. Anti-IKBKG/NEMO antibodies were purchased from Sigma Aldrich (SAB1404591) and Millipore (05-631).
Cell lines used in this study HEK293T (CRL-3216), HeLa (CCL-2), CaCo-2 (HTB-37) and U2OS (HTB-96) were purchased from ATCC. Parental cell lines were tested and determined to be mycoplasma free.

de Jong M.F., Liu Z., Chen D, & Alto N.M. (2016). Shigella flexneri suppresses NF-kB activation by inhibiting linear ubiquitin chain ligation. Nature microbiology, 1(7), 16084.

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Comprehensive Cell Line Cultivation Protocol

Cited 1 time

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MDA-MB-231 (HTB-26), HCT-116(CCL-247), LnCaP (Clone FGC, CRL-1740), RH30 (CRL-2061), SJSA (CRL-2098), HOS (CRL-1543), SAOS2 (HTB-85) and U2-OS (HTB-96) cells were purchased from ATCC (Manassas, VA). MCF-7 (86012803) was purchased from Sigma Aldrich (St. Louis, MO). CF-1 is a cell culture isolated from an 18 month old male presenting with alveolar rhabdomyosarcoma (22 ). PCB-204 is a cell culture isolated from the high-grade, extra-skeletal osteosarcoma of a 33 year old male (23 ). MCF-7 and MDA-MB-231 were cultured in DMEM media (Thermo Fisher, MA, 11995073) supplemented with 10% fetal bovine serum (Thermo Fisher, 10437036) and 1% penicillin/ streptomycin (Thermo Fisher, 15140112). All other human cells were cultured in complete RPMI media (Thermo Fisher, 11875119) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. 61323 is an osteosarcoma cell culture isolated from a tumor arising from an Osx1-Cre, Trp53−/−, Rb1−/− genetically engineered mouse as previously characterized (24 (link)). 61323 was cultured in DMEM media supplemented with 10% fetal bovine serum and 1% penicillin/ streptomycin. All human cell cultures were authenticated using short tandem repeat analysis performed by the University of Arizona Genetics Core (Tuscon, AZ).

Cleary M.M., Bharathy N., Abraham J., Rudzinski E., Michalek J.E, & Keller C. (2021). Interleukin-4 receptor inhibition targeting metastasis independent of macrophages. Molecular cancer therapeutics, 20(5), 906-914.

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Characterization of Cell Lines for Research

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HEK293 (CRL-1573), HepG2 (HB-8065), and U2OS (HTB-96) cells were purchased from ATCC. Human skin fibroblasts HSFs (F2-S) and primary MEFs were prepared as described previously (Liu et al., 2005 (link)). Immortalized Atm-/-; p53-/- and Sirt6-/- MEFs were provided as a kind gift from Dr. Yosef Shiloh (Tel Aviv University, Israel) and Dr. Raul Mostoslavsky (Massachusetts General Hospital Cancer center, USA), respectively. These cell lines were authenticated by short tandem repeat (STR) profile analysis and genotyping, and were mycoplasma free. Cells were cultured in Gibco DMEM (Life Technologies, USA) with 10% fetal bovine serum (FBS), 100 U/ml penicillin and streptomycin (P/S) at 37°C in 5% CO₂ and atmospheric oxygen conditions. For CQ experiments, cells were maintained in the medium containing 1 μM chloroquine for 12 hr, and then grown in new fresh medium for 48 hr.

Qian M., Liu Z., Peng L., Tang X., Meng F., Ao Y., Zhou M., Wang M., Cao X., Qin B., Wang Z., Zhou Z., Wang G., Gao Z., Xu J, & Liu B. (2018). Boosting ATM activity alleviates aging and extends lifespan in a mouse model of progeria. eLife, 7, e34836.

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Generation of Isogenic Sarcoma Cell Lines

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To generate Atrx isogenic mouse sarcoma cell lines, pSP Cas9(BB)-2A-GFP (PX458) with Atrx gRNA or vector control were transfected into newly derived primary soft tissue sarcoma cell lines (KP) using the TransIT-LT1 transfection reagent (Mirus). Cell lines from transfected single-cell clones were then screened for loss of ATRX protein expression by Western blot. Antibodies used for Western blot and immunofluorescence are listed in Supplemental Table 1. To generate human sarcoma cell lines, 143B cells were purchased (ATCC, CRL8303) and an identical procedure was performed, except the gRNA in the PX458 vector was targeted to the human ATRX gene. Passaging of sarcoma cell lines was performed in DMEM (Thermo Fisher Scientific) supplemented with 10% FBS (Thermo Fisher Scientific), 1% Pen-Strep (Thermo Fisher Scientific), and 1% L-glutamine (Thermo Fisher Scientific). Cell lines were incubated at 37°C with 5% CO₂ in a humidified cell culture incubator. U2OS (HTB-96) and 143B (CRL-8303) cell lines were purchased from ATCC.

Floyd W., Pierpoint M., Su C., Patel R., Luo L., Deland K., Wisdom A.J., Zhu D., Ma Y., DeWitt S.B., Williams N.T., Lazarides A.L., Somarelli J.A., Corcoran D.L., Eward W.C., Cardona D.M, & Kirsch D.G. (2023). Atrx deletion impairs CGAS/STING signaling and increases sarcoma response to radiation and oncolytic herpesvirus. The Journal of Clinical Investigation, 133(13), e149310.

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Comprehensive Antibody Validation Protocol

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de Jong M.F., Liu Z., Chen D, & Alto N.M. (2016). Shigella flexneri suppresses NF-kB activation by inhibiting linear ubiquitin chain ligation. Nature microbiology, 1(7), 16084.

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Inducible Expression of Fluorescent Proteins in Cell Lines

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MEFs were isolated from 13.5-dpc (day post coitum) embryos of wild-type mice. MEFs were stocked and cultured for maximum of five passages. HeLa (CCL-2) and U2OS (HTB-96) cell lines were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA). HeLa tet-on cell line was purchased from Clontech (Seoul, Rep. of Korea). HeLa, HeLa tet-on and MEFs were cultured in Dulbecco's modified Eagle's medium (Dulbecco's modified Eagle's medium (DMEM); WelGENE, Gyeongsan, Rep. of Korea) containing 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA). U2OS cells were cultured in RPMI 1640 (WelGENE) containing 10% FBS. To generate HeLa cells inducibly expressing GFP or GFP-BEX4 proteins, HeLa tet-on cells were transfected with pTRE2hyg-GFP or pTRE2hyg-GFP-BEX4. Colonies showing resistance to hygromycin (20 μg/ml) were clonally isolated. GFP and GFP-BEX4 proteins were induced by treating cells with doxycycline (2 μg/ml) for 24 h.

Lee J.K., Lee J., Go H., Lee C.G., Kim S., Kim H.S., Cho H., Choi K.S., Ha G.H, & Lee C.W. (2016). Oncogenic microtubule hyperacetylation through BEX4-mediated sirtuin 2 inhibition. Cell Death & Disease, 7(8), e2336-.

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Cell Culture Protocols for Standard Cell Lines

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HeLa (CCL2, ATCC), U2OS (HTB-96, ATCC) and HEK293T (CRL3216, ATCC) cell lines were purchased from ATCC (American Type Culture Collection, VA, USA). Short tandem repeat (STR) testing was performed to confirm cell identity. These cells were cultured in Dulbecco’s Modified Eagle Medium (C11995500BT, GIBCO) supplemented with 10% fetal bovine serum (10270-106, GIBCO) and incubated at 37 °C and 5% CO₂ in a constant temperature incubator. Cell passaging was performed at a 1:3 split ratio when the cells reached 90% confluence.

Cui Z., Tian R., Huang Z., Jin Z., Li L., Liu J., Huang Z., Xie H., Liu D., Mo H., Zhou R., Lang B., Meng B., Weng H, & Hu Z. (2022). FrCas9 is a CRISPR/Cas9 system with high editing efficiency and fidelity. Nature Communications, 13, 1425.

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Plasmid and siRNA Transfection in HeLa, U-2 OS, and MRC-5 Cells

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HeLa cells (CCL-2) and U-2 OS (HTB-96) cells were purchased from ATCC. HeLa and U-2 OS cells were maintained in Eagle’s minimal essential medium (ATCC) and McCoy’s 5A medium (ATCC), respectively, with 10% FBS (Corning) and 1× penicillin/streptomycin solution (Corning). MRC-5 fibroblasts were kindly provided by Dr. Evan Reid and cultured in DMEM (Thermo Fisher Scientific) supplemented with 10% FBS and 1× penicillin/streptomycin solution. For imaging experiments, DNA plasmids (15–50 ng) and siRNAs (∼20 nM) were transfected into HeLa cells with TransIT-LT1 reagent for 16–20 h and TransIT-TKO reagent for 64–68 h, respectively (Muris). For mEmerald-SKL or mCherry-SKL and siRNA cotransfection, HeLa cells were transfected simultaneously with 10 ng plasmid and 20 nM siRNA using both TransIT-LT1 and TransIT-TKO following the manufacturer’s instructions. For plasmids and siRNA transfection in Fig. 5, HeLa cells were first transfected with siRNA using TransIT-TKO for 48 h and subsequently transfected with 15 ng mEmerald-SKL and 50 ng mApple-M1 Spastin using TransIT-X2 for ∼16 h. U-2 OS and MRC-5 cells were transfected with 15–50 ng DNA plasmids using TransIT-X2.

Chang C.L., Weigel A.V., Ioannou M.S., Pasolli H.A., Xu C.S., Peale D.R., Shtengel G., Freeman M., Hess H.F., Blackstone C, & Lippincott-Schwartz J. (2019). Spastin tethers lipid droplets to peroxisomes and directs fatty acid trafficking through ESCRT-III. The Journal of Cell Biology, 218(8), 2583-2599.

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