Tetracycline free fetal bovine serum

Manufactured by Takara Bio

Sourced in United States

Tetracycline-free fetal bovine serum is a cell culture supplement derived from the blood of fetal bovine (cow) fetuses. It provides a source of nutrients, growth factors, and other components necessary for the cultivation of various cell types in vitro.

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13 protocols using tetracycline free fetal bovine serum

HA-tagged TFE3 Fusion Protein Expression

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HK2 cell lines, which express HA-PRCC-TFE3, HA-SFPQ-TFE3, or HA-NONO-TFE3 doxycycline-dependently, were established by two sequential lentiviral transductions using the full-length rtTA3(Tet-on) gene (Takara) and the HA-PRCC-TFE3, HA-SFPQ-TFE3, or HA-NONO-TFE3 genes under control of the TRE-tight promoter. Cells were cultured in Advanced DMEM/F-12 with 1.5% Tetracycline-Free Fetal Bovine Serum (Takara) and selection antibiotics, 2 µg/mL Blasticidin S, and 0.8 µg/mL Puromycin. For gene expression profiling, HK2 lines were cultured with or without 200 ng/mL doxycycline. Total RNA was isolated from cells using TRIzol reagent (Invitrogen). cDNA preparation and hybridization were performed according to the manufacturer's protocol (Affymetrix). Affymetrix GeneChip Human Genome U133 Plus 2.0 arrays were applied. RMA-based data normalization and subsequent data analysis, including PCA and ANOVA, were performed with Partek Genomics Suite 6.6.

Horiguchi H., Kadomatsu T., Kurahashi R., Hara C., Miyata K., Baba M., Osumi H., Terada K., Araki K., Takai T., Kamba T., Linehan W.M., Moroishi T, & Oike Y. (2019). Dual functions of angiopoietin-like protein 2 signaling in tumor progression and anti-tumor immunity. Genes & Development, 33(23-24), 1641-1656.

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Engineered HEK293 Cell Lines

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HEK293 parental Ub-replacement cells were generated following previous protocol provided by Z. J. Chen (University of Texas Southwestern Medical Center, Dallas, TX). All Ub replacement cells were made as previously described (25).The HEK293 cell line stably expressing Ub shRNA and Ub WT, T66A, and T66E mutants was maintained in DMEM containing 10% tetracycline-free fetal bovine serum (Takara). DOX was added to the medium when indicated.

Deng M., Lin J., Nowsheen S., Liu T., Zhao Y., Villalta P.W., Sicard D., Tschumperlin D.J., Lee S., Kim J, & Lou Z. (2020). Extracellular matrix stiffness determines DNA repair efficiency and cellular sensitivity to genotoxic agents. Science Advances, 6(37), eabb2630.

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Cell Line Maintenance and Antibody Sourcing

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HEK-293FT (Thermo Fisher Scientific, Waltham, MA), HeLa CD4+ (Maddon et al., 1986 (link)) and TZM-bl (Platt et al., 2009 (link)) (NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH) cells were maintained in Dulbecco's Modified Eagle's medium (DMEM) medium containing 10% fetal bovine serum and 500 μg/ml G418 (for HeLa CD4+ cells). Tet-On HeLa-GADD34/DOX (Dr. Stefan Marciniak, University of Cambridge, Cambridge, UK) cells were maintained in DMEM medium containing 10% tetracycline-free fetal bovine serum (Takara Bio, Mountain View, CA), 200 μg /ml of G418, and 200 uM of hygromycin. MT2 cells (Harada et al., 1985 ) (NIH AIDS Reagent Program) and Jurkat (ATCC, Manassas, VA) were maintained in RPMI 1640 medium containing 10% fetal bovine serum. Antibodies to α-tubulin, GADD34 specific for amino acids 307-572, GADD34 specific for full length protein, FLAG-tag, and HIV-1 CA-p24 were from Santa Cruz Biotechnology (Dallas, Texas), Proteintech (Rosemont, IL), ThermoFisher ( Waltham, MA), OriGene (Rockville, MD), and the NIH AIDS Reagent Program, respectively. Doxycycline hydrochloride (DOX), hygromycin, and G418 were from Millipore Sigma (St. Louis, MO).

Ishaq M., Marshall H, & Natarajan V. (2019). GADD34 attenuates HIV-1 replication by viral 5'-UTR TAR RNA-mediated translational inhibition. Virology, 540, 119-131.

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SNCA Expression in SH-SY5Y Cells

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SNCA^WT SH-SY5Y cells and SNCA^A53T SH-SY5Y cells were established using Lenti-X™ Tet-On® 3G inducible expression system and SH-SY5Y cells according to user manual. Doxycycline (Dox; Clontech Laboratories, CA, USA) was used to induce the expression of SNCA^WT and SNCA^A53T. Cells were routinely grown in Dulbecco's Modified Eagle's Medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% tetracycline-free fetal bovine serum (Clontech Laboratories, CA, USA) and cultured at 37°C under humidified 5% CO2 atmosphere. When cells were subcultured once attaining 70-80% confluency, MANF (PeproTech, State of NJ, USA) and Dox were added for 24 or 48 h, respectively.

Zhang J.X., Tong W.F., Jiang M., Zhou K.G., Xiang X.R., He Y.J., Zhang Z.Y., Guan Q, & Jin L.J. (2022). MANF Inhibits α-Synuclein Accumulation through Activation of Autophagic Pathways. Oxidative Medicine and Cellular Longevity, 2022, 7925686.

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HeLa and DLD-1 Cell Culture and Transfection

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HeLa and DLD-1 cells were maintained at 37°C in a 5% CO₂ atmosphere in DMEM (Thermo Fischer Scientific, Waltham, MA) supplemented with 10% tetracycline-free fetal bovine serum (Clontech, Mountain View, CA), 100 U/ml penicillin, 100 U/ml streptomycin, and 2 mM l-glutamine. For immunofluorescence, cells were seeded on 12-mm poly-l-lysine–coated coverslips in 12-well plates 24 h before transfection with siRNAs. For live-cell imaging experiments, cells were seeded in a 35-mm glass-bottom dish coated with poly-d-lysine (MatTek, Ashland, MA). Cells were transfected with siRNAs targeting Spindly and the CENP-E 3′ UTR as described previously (Gassmann et al., 2010 (link); Kim et al., 2010 (link)). For immunofluorescence of HeLa Flp-In T-Rex cells, transgene expression was induced with 0.2 μg/ml tetracycline 24 h posttransfection, and cells were fixed 20–24 h later. For live-cell imaging of HeLa Flp-In T-Rex cells and for the immunoblot shown in Figure 5A, transgene expression was induced 22 h after transfection, and time-lapse imaging experiments were started 8 h later.

Holland A.J., Reis R.M., Niessen S., Pereira C., Andres D.A., Spielmann H.P., Cleveland D.W., Desai A, & Gassmann R. (2015). Preventing farnesylation of the dynein adaptor Spindly contributes to the mitotic defects caused by farnesyltransferase inhibitors. Molecular Biology of the Cell, 26(10), 1845-1856.

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Inducible BRCA2 Silencing in Cancer Cell Lines

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Human non-small cell lung carcinoma H1299 cells and human invasive ductal breast cancer MDA-MB-231 cells, wild type (American Type Culture Collection) or carrying a doxycycline (DOX)-inducible BRCA2 shRNA^{4 (link)}, were cultivated in monolayers in DMEM medium (Sigma) supplemented with 10% tetracycline free fetal bovine serum (Clontech). For induction of shBRCA2, 2 µg/mL DOX (D9891, Sigma) was added to growth medium. Human colorectal adenocarcinoma DLD1 cells, parental and BRCA2-mutated (Horizon Discovery^{3 (link)}), were cultivated in monolayers in DMEM medium (Sigma) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and 1% penicillin/streptomycin (Sigma).

Reisländer T., Lombardi E.P., Groelly F.J., Miar A., Porru M., Di Vito S., Wright B., Lockstone H., Biroccio A., Harris A., Londoño-Vallejo A, & Tarsounas M. (2019). BRCA2 abrogation triggers innate immune responses potentiated by treatment with PARP inhibitors. Nature Communications, 10, 3143.

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Cell Culture Protocol for Multiple Cell Lines

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HT-29, 293T, DLD-1, and HCT-8 cells were obtained from the American Type Culture Collection (ATCC) and cultured according to their recommendations. The 5E clone of MCF10A cells was cultured as described previously (119 (link)). AC16 cells (110 (link)) were purchased from M. Davidson (Columbia University) and cultured in Dulbecco’s modified Eagle’s medium/F-12 medium (Life Technologies) with 12.5% tetracycline-free fetal bovine serum (Clontech) and penicillin-streptomycin (Gibco).

Chitforoushzadeh Z., Ye Z., Sheng Z., LaRue S., Fry R.C., Lauffenburger D.A, & Janes K.A. (2016). TNF-insulin crosstalk at the transcription factor GATA6 is revealed by a model that links signaling and transcriptomic data tensors. Science signaling, 9(431), ra59.

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Chemically Inducing ER Stress

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Tunicamycin and thapsigargin (Sigma) were used at the concentrations specified in the figure legends. For chemical ER stress experiments involving inducible overexpression or knockdown of Wfs1, RPMI 1640 media (ThermoFisher Scientific) was supplemented with tetracycline-free fetal bovine serum (Clontech) and β-mercaptoethanol (Sigma). For glucotoxic ER stress experiments, RPMI 1640 no glucose media (ThermoFisher Scientific, cat no 11879020) was supplemented with D-glucose (Gibo) as specified in figure legends.

Abreu D., Asada R., Revilla J.M., Lavagnino Z., Kries K., Piston D.W, & Urano F. (2020). Wolfram syndrome 1 gene regulates pathways maintaining beta cell health and survival. Laboratory investigation; a journal of technical methods and pathology, 100(6), 849-862.

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Inducible Lentiviral Transduction of MKK7-JNK1 Fusion

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HEK293T cells (American Type Culture Collection Cat # CRL-3216) were transfected with 7.5μg each of the packaging plasmids pMD2.G (Addgene plasmid #12259 (Naldini et al. 1996 (link))) and psPAX2 (Addgene plasmid # 12260 (Naldini et al. 1996 (link))) plus 10μg of pSLIK-Hygro or pSLIK-Flag-MKK7β2-Jnk1α1-Hygro using Lipofectamine 2000 (Life Technologies). The culture supernatant was collected at 24 h post-transfection and filtered (0.45 μm). Primary epithelial cells were transduced (×2) with the lentivirus plus polybrene (8 μg/ml). The transduced epithelial cells were selected at 48 h post-infection using medium supplemented with 8 μg/ml hygromicin (Life Technologies). The cells were maintained in selection medium with tetracycline-free fetal bovine serum (Clontech). To induce expression of the MKK7-JNK1 fusion protein, the cells were treated with 1 μg/ml doxycycline (24 h).

Girnius N, & Davis R.J. (2017). JNK promotes epithelial cell anoikis by transcriptional and post-translational regulation of BH3-only proteins. Cell reports, 21(7), 1910-1921.

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Chemically Inducing ER Stress

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