Atcc htb 14

Manufactured by American Type Culture Collection

Sourced in United States, France

ATCC® HTB-14™ is a cell line derived from a human glioblastoma. It is a commonly used model for the study of brain cancer.

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U-87 MG (ATCC HTB-14) U-87 MG ATCC® HTB-14™ Homo sapiens brain U-87MG ATCC® HTB-14™ Homo sapiens brain Likely glioblastomas U87 (ATCC HTB-14) HTB-14TM HTB-14

25 protocols using atcc htb 14

Carboxymethylcellulose-Based Doxorubicin Delivery System

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Carboxymethylcellulose sodium salt (CMC, DS = 0.7, M_w = 250 kDa; viscosity = 735 cps at 2% in H₂O, at 25 ºC), L-cysteine hydrochloride (Cys, HSCH₂CH (NH₂)COOH · HCl, ≥ 98%), and doxorubicin hydrochloride (DOX, C₂₇H₂₉NO₁₁ ⋅ HCl, ≥ 98.0%) were purchased from Sigma-Aldrich (USA). KLA (LAKLAKKLAKLAK) and KLAR7 (RRRRRRRKLAKLAKKLAKLAK) peptides were purchased from GenScript (USA). All of the other materials used in this research were detailed in the Supplementary Material.
Human embryonic kidney cells (HEK 293T, American Type Culture Collection - ATCC CRL-1573) were provided by the Federal University of Minas Gerais/UFMG. Human brain likely glioblastoma cells (U-87 MG, ATCC HTB-14) were supplied from Brazilian Cell Repository (Banco de Células do Rio de Janeiro: BCRJ, Brazil; cell line authentication molecular technique, Short Tandem Repeat (STR) DNA; quality assurance validation by international standard NBR ISO/IEC 17025:2005).

Carvalho I.C., Mansur A.A., Carvalho S.M, & Mansur H.S. (2021). Nanotheranostics through Mitochondria-targeted Delivery with Fluorescent Peptidomimetic Nanohybrids for Apoptosis Induction of Brain Cancer Cells. Nanotheranostics, 5(2), 213-239.

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Glioblastoma Cell Lines for Cancer Research

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Human malignant glioma cell lines U87MG (ATCC^® HTB-14™) and DBTRG-05MG (ATCC^® CRL-2020™) were purchased from American Type Culture Collection (ATCC) for this study. U87MG is categorized as glioblastoma; astrocytoma; classified as grade IV by ATCC. The DBTRG-O5MG (Denver Brain Tumor Research Group 05) cell line was established from the tumor tissue of a GBM patient who had been treated with local brain irradiation and multidrug chemotherapy. U87MG were grown in complete Dulbecco's modified Eagle medium (DMEM) (GIBCO) and Roswell Park Memorial Institute (RPMI) (Sigma-Aldrich), supplemented with 10 % fetal bovine serum (GIBCO) in an incubator with 5 % CO2, respectively. Ibrutinib (PCI-32765) and temozolomide stocks (SelleckChem, Taiwan) were dissolved in DMSO. The final concentration of DMSO in the culture medium should be adjusted to be below 0.01 % and not affect the cell viability and the expression of the proteins.

Wei L., Su Y.K., Lin C.M., Chao T.Y., Huang S.P., Huynh T.T., Jan H.J., Whang-Peng J., Chiou J.F., Wu A.T, & Hsiao M. (2016). Preclinical investigation of ibrutinib, a Bruton's kinase tyrosine (Btk) inhibitor, in suppressing glioma tumorigenesis and stem cell phenotypes. Oncotarget, 7(43), 69961-69975.

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Optimized Tumor Cell Lines for Angiogenesis

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Rat 9L or human U87 glioblastoma cells were used to create the tumor onplants. Rat 9L cells were obtained from Dr. Martin Friedlander and Dr. Faith Barnett at the Scripps Research Institute. These cells were a frozen aliquot from those used in previous publications [28 (link), 36 (link)]. Human U87 glioblastoma cells were obtained from ATCC (ATCC HTB-14), along with human cell-line authentication. Low-passage aliquots were frozen in liquid nitrogen. Cells were grown and maintained in high glucose DMEM media with L-glutamine (ThermoFisher cat# 11965092) and passaged according to standard protocol, ensuring that confluency never exceeded 80% and cells never exceeded passage ten to prevent unwanted cellular changes. Twenty-four hours prior to use in the study, cell media was replaced with serum-free, low glucose DMEM media (ThermoFisher cat# 11885076) to activate the tumor cells towards a more energy depleted state, thus optimizing pro-angiogenic activity.

Dorrell M.I., Kast-Woelbern H.R., Botts R.T., Bravo S.A., Tremblay J.R., Giles S., Wada J.F., Alexander M., Garcia E., Villegas G., Booth C.B., Purington K.J., Everett H.M., Siles E.N., Wheelock M., Silva J.A., Fortin B.M., Lowey C.A., Hale A.L., Kurz T.L., Rusing J.C., Goral D.M., Thompson P., Johnson A.M., Elson D.J., Tadros R., Gillette C.E., Coopwood C., Rausch A.L, & Snowbarger J.M. (2021). A novel method of screening combinations of angiostatics identifies bevacizumab and temsirolimus as synergistic inhibitors of glioma-induced angiogenesis. PLoS ONE, 16(6), e0252233.

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Culturing U-87 MG Glioblastoma Cells

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U-87 MG human glioblastoma cell line was purchased from American Type Culture Collection (ATCC® HTB-14™; ATCC, Manassas, VA, USA). U-87 MG cells were grown in Minimum Essential Medium (Life Technologies, Paisley, UK) supplemented with FBS (10% ^v/_v; Labtech International Ltd, Uckfield, East Sussex, UK); sodium pyruvate (1 mM; Life Technologies) and L-Glutamine (2 mM; Life Technologies). Cells were maintained in culture (37°C in 5% CO₂) for up to 14 days (splitting every 2–3 days when 75–80% confluence was reached in the 75 cm² tissue culture flask) before they were used for tumour implantation.

Fisusi F.A., Siew A., Chooi K.W., Okubanjo O., Garrett N., Lalatsa K., Serrano D., Summers I., Moger J., Stapleton P., Satchi-Fainaro R., Schätzlein A.G, & Uchegbu I.F. (2016). Lomustine Nanoparticles Enable Both Bone Marrow Sparing and High Brain Drug Levels – A Strategy for Brain Cancer Treatments. Pharmaceutical Research, 33, 1289-1303.

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Investigating SEMA3B-AS1 and miR-195 in GBM

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Human GBM cell lines U87 and U251 (ATCC® HTB14™, ATCC) were used. DMEM medium supplemented with 10% FBS and 100 U/ml penicillin/streptomycin was used to culture cells at 37°C with 5% CO₂. SEMA3B-AS1 and cyclin D1 expression vectors (pcDNA3.1) were constructed by Sangon (Shanghai, China). SEMA3B-AS1 siRNA and NC siRNA were synthesized by Invitrogen (Shanghai, China). MiR-195 mimic and inhibitor as well as negative control (NC) were purchased from Sigma-Aldrich (USA). All transient cell transfections were performed using lipofectamine 2000 transfection reagent (Sigma-Aldrich, USA) to transfect 10 nM vectors, 30 nM miRNA, 30 nM siRNA or 30 nM inhibitor into 10⁵ cells. Transfection with empty pcDNA3.1 vector, negative control miRNA or inhibitor negative control was used as negative control (NC). Cells without transfection were used as the Control (C) cells.

Liu K., Deng Y., Yang Y., Wang H, & Zhou P. (2022). MicorRNA-195 links long non-coding RNA SEMA3B antisense RNA 1 (head to head) and cyclin D1 to regulate the proliferation of glioblastoma cells. Bioengineered, 13(4), 8798-8805.

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Characterization of Glioblastoma Cell Lines

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M7 (NRAS, SV-40LgT) and OL61 (shp53/PDGFB/NRAS ) were a gift from Dr. John Ohlfest, Department of Pediatrics, University of Minnesota (3 (link)); they were not further authenticated by us. U87MG cells (ATCC® HTB-14™) were purchased from ATCC and were not further authenticated by us. U251 cells were a gift from Dr John L. Darling, Institute of Neurology, London, England; they have not been further authenticated by us. HF2303 cells were provided by Dr. Tom Mikkelsen (7 (link)) (Department of Neurology, Henry Ford Hospital, Detroit, MI) and were not further authenticated by us. M1, M4, M5 were generated as described (2 ) with NRAS and SV-40 LgT, characterized in this manuscript and were not further authenticated by us. Culture conditions are detailed in the Supplementary Methods.
In vitro analysis of apoptosis, proliferation and cell cycle progression, Western Blot Analysis and Quantitative Real-Time PCR were performed using standard methods. Experimental details, antibodies and primers used are presented in the Supplementary Methods.
Statistical Analysis was performed using GraphPad Software with either ANOVA or t-test as specified in the figure legends. Kaplan-Meier survival curves were analyzed using the Mantel-Cox log-rank test.

Calinescu A.A., Yadav V., Carballo E., Kadiyala P., Tran D., Zamler D.B., Doherty R., Srikanth M., Lowenstein P.R, & Castro M.G. (2016). Survival and proliferation of neural progenitor derived glioblastomas under hypoxic stress is controlled by a CXCL12/CXCR4 autocrine positive feedback mechanism. Clinical cancer research : an official journal of the American Association for Cancer Research, 23(5), 1250-1262.

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Culturing Glioma, Colorectal, and EGFR-Overexpressing Cell Lines

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EGFR-overexpressing cell lines, glioma cell lines U87 MG (ATCC HTB-14) and C6 (ATCC CCL-107), and colorectal adenocarcinoma cell line HT-29 (ATCC^® HTB-38) were obtained from ATCC culture collection (Virginia City, NV, USA). F98 rat glioma cells, histologically characterized as an anaplastic astrocytoma, were a kind gift from Dr. Rolf Barth (Dept. of Pathology, The Ohio State University, Columbus, OH, USA). Cells were cultured in Dulbecco modified Eagle’s medium high glucose (4.5 g/L) with stable glutamine (3.97 mM) (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (i-FBS) and 1% of a solution containing penicillin-streptomycin (PS-B) at 37 °C in a humid atmosphere containing 5% CO₂ concentration. Culturing materials were purchased from Capricorn Scientific. In order to obtain i-FBS, original FBS was heated at 60 °C for 30 min.

Couto M., Alamón C., García M.F., Kovacs M., Trias E., Nievas S., Pozzi E., Curotto P., Thorp S., Dagrosa M.A., Teixidor F., Viñas C, & Cerecetto H. (2020). Closo-Carboranyl- and Metallacarboranyl [1,2,3]triazolyl-Decorated Lapatinib-Scaffold for Cancer Therapy Combining Tyrosine Kinase Inhibition and Boron Neutron Capture Therapy. Cells, 9(6), 1408.

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Glioma Cell Line Characterization and Transfection

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Glioma cell lines U87 MG (glioblastoma of unknown origin, ATCC^® HTB-14; ATCC), LN229 (ATCC^® CRL-2611), A172 (ATCC^® CRL-1620), U373 MG (ATCC^® HTB-17), U251 (U251 MG; cat. no. YS448C; YaJi Biological), human normal astrocytes NHA (cat. no. YS2144C; YaJi Biological) and 293T cells (cat. no. YS005C; YaJi Biological) were cultured and preserved in DMEM (GIBCO-BRL; Thermo Fisher Scientific, Inc.) supplemented with 100 U/ml penicillin, 10% fetal bovine serum, and 100 mg/ml streptomycin (Beyotime Institute of Biotechnology) in a humidified atmosphere containing 5% CO₂ at 37°C. STR profiling analysis was performed for the authentication of cell lines.
As per the guidance of the manufacturer (Shanghai GenePharma Co., Ltd.), LINC00665 overexpression (OE) plasmid/small interfering (si)RNA and microRNA (miR)-34a-5p mimics/inhibitor were used for transfection assays with Lipofectamine 2000 Reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Cells grown to approximately 50–60% confluence in culture dishes were used for transfection. Transfection was performed in serum-free medium for one day.

Dai Y., Zhang Y., Hao M, & Zhu R. (2021). LINC00665 functions as a competitive endogenous RNA to regulate AGTR1 expression by sponging miR-34a-5p in glioma. Oncology Reports, 45(3), 1202-1212.

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Glioblastoma and Bone Marrow Stromal Cell Culture

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For cell culturing [27 (link)], the human glioblastoma U87 MG (ATCC^® HTB-14™) and bone marrow stromal Hs5 (ATCC^® CRL-11882™) cell lines were used from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Life Technologies, Houston, TX, USA) and 1% antibiotic-antimycotic mixture containing penicillin and streptomycin (Life Technologies, Houston, TX, USA) at 37 °C under 5% CO₂ and 95% humidity in an INCOMED153 (Memmert GmbH & Co. KG, Schwabach, Germany).

Szczepaniak J., Sosnowska M., Wierzbicki M., Witkowska-Pilaszewicz O., Strojny-Cieslak B., Jagiello J., Fraczek W., Kusmierz M, & Grodzik M. (2022). Reduced Graphene Oxide Modulates the FAK-Dependent Signaling Pathway in Glioblastoma Multiforme Cells In Vitro. Materials, 15(17), 5843.

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Culturing Human Glioblastoma and Breast Cancer Cells

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Human glioblastoma cells -U87(ATCC® HTB14™) were obtained from ATCC, MCF10CA1h cells [35] were received from the Barbara Ann Karmanos Cancer Institute (Detroit, MI, USA), and they were cultured as previously described. Briefly, the U87 cells were cultured in DMEM -Dulbecco's Modified Eagle Medium (ThermoFisher Scientific, 11995065) that was supplemented with 10% fetal bovine serum (FBS) (ThermoFisher Scientific, 11995065) and 50 U/mL penicillin and 50 µg/ml streptomycin (Thermo Fisher, 15070-063). MCF10CA1h cells were cultured in the complete medium: DMEM/F12 (11330-032, Thermo Fisher Scientific), 5% horse serum (16050-122, Thermo Fisher Scientific), 5 ng/ml EGF (AF-100-15-1MG, Peprotech), 0.5 mg/ml Hydrocortisone (H0888-1G, Sigma-Aldrich), 100 ng/ml Cholera toxin (C8052-2mg, Sigma-Aldrich), 10 µg/ml insulin (I1882-100MG, Sigma-Aldrich) and 1x penicillin/streptomycin solution (15070-063, Thermo Fisher Scientific). Cells were cultured at 37˚C, in 5% CO2. Cell propagation was performed by detaching adherent cells using Trypsin (0.25% for U87 cells (ThermoFisher Scientific, 80-2101) and 0.05% for MCF10CA1h cells (25-052-Cl, Corning)) as previously described. All experiments were performed using cells with passage number less than 19. Cell medium was changed every 2-3 days.

Nikolić M., Scarcelli G., & Tanner K. (2022). Multimodal microscale mechanical mapping of cancer cells in complex microenvironments.

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