Tet system approved fbs

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Tet System Approved FBS is a high-quality fetal bovine serum (FBS) that has been tested and approved for use in Tet-based expression systems. It is designed to support the growth and maintenance of cells in culture while ensuring the reliable performance of Tet-regulated gene expression.

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Tet-approved FBS (24 citations)

88 protocols using tet system approved fbs

Cell Culture Protocols for CHO and C2C12 Lines

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CHO-K1 (Hamster cells, RRID:CVCL_0214, ATCC Catalog No. CCL-61) or
CHO- TREx (RRID:CVCL_D586, Invitrogen) cells and their derivatives were
grown on tissue-culture grade plastic plates (Thermo Scientific) in Alpha
MEM Earle’s Salts (Life Technologies), supplemented with 10% Tet
System Approved FBS (ClonTech), 100 U/ml penicillin, 100 ug/ml streptomycin,
0.292 mg/ml L-glutamine (GIBCO).
C2C12 cells (Mouse cells, RRID:CVCL_0188, ATCC Catalog No. CRL-1772)
were grown in DMEM (Life Technologies), supplemented with 20% Tet System
Approved FBS (ClonTech), 100 U/ml penicillin, 100 ug/ml streptomycin, 0.584
mg/ml L-glutamine (GIBCO). C2C12 media was used for CHO-K1 + C2C12
co-culture assays (Figure
S4). All cells were grown at 37° C in 5% CO₂ in
a humidified atmosphere. Cells were passaged every 2–3 days,
depending on confluency, using 0.05% or 0.25% Trypsin-EDTA (Life
Technologies).

Nandagopal N., Santat L.A., LeBon L., Sprinzak D., Bronner M.E, & Elowitz M.B. (2018). Dynamic Ligand Discrimination in the Notch Signaling Pathway. Cell, 172(4), 869-880.e19.

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Cell Culture Protocols for CHO and C2C12 Lines

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Nandagopal N., Santat L.A., LeBon L., Sprinzak D., Bronner M.E, & Elowitz M.B. (2018). Dynamic Ligand Discrimination in the Notch Signaling Pathway. Cell, 172(4), 869-880.e19.

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Establishment and Maintenance of NPC Cell Lines

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Primary NPC fresh tumor tissues and fresh non-cancer nasopharyngitis tissues were acquired from the Tumor Tissue Bank of Sun Yat-Sen University Cancer Center, and the detailed relevant information has been described previously.^{32 (link)} This study was approved by the Institute Research Medical Ethics Committee of Sun Yat-Sen University Cancer Center, and written informed consent was obtained from all patients. NPC cell lines were maintained in RPMI 1640 medium (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) in a humidified 5% CO₂ incubator at 37 °C. Primary NPEC1 cultures and immortalized NPEC1 cells induced by Bmi-1 were established as described previously^{51 (link)} and grown in keratinocyte/serum-free medium (Invitrogen, Carlsbad, CA, USA). Tet-Off-inducible advanced cell lines were maintained in RPMI 1640 medium supplemented with 10% Tet-System Approved FBS (Clontech, Mountain View, CA, USA) with both G418 (400 μg/ml) and doxycycline (200 ng/ml). Tet-Off-inducible RBM24 stable cells were maintained in RPMI 1640 medium supplemented with 10% Tet-System Approved FBS (Clontech) with G418 (400 μg/ml), puromycin (0.5 μg/ml) and doxycycline (200 ng/ml).

Hua W.F., Zhong Q., Xia T.L., Chen Q., Zhang M.Y., Zhou A.J., Tu Z.W., Qu C., Li M.Z., Xia Y.F., Wang H.Y., Xie D., Claret F.X., Song E.W, & Zeng M.S. (2016). RBM24 suppresses cancer progression by upregulating miR-25 to target MALAT1 in nasopharyngeal carcinoma. Cell Death & Disease, 7(9), e2352-.

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Comprehensive Antibody Validation for GLI2 and Hedgehog Pathway

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Antibodies against GLI2 H-300 (sc-28674) and GAPDH (sc-32233) were purchased from Santa Cruz Biotechnology; GLI3 (AF3690) from R and D Systems; SHH (ab53281), SMO (ab38686) and S100A2 (ab109494) from Abcam; GLI2 (18989–1-AP), KRAS (12063–1-AP), Tubulin (66031–1-Ig), E-Cadherin (20874–1-AP), and ESRP1 (21045–1-AP) from Proteintech; GLI1 (2534), FLAG (2368), ZEB1 (3396), Vimentin (5741), YAP1 (4912), N-Cadherin (4061), GATA6 (5851) from Cell Signaling Technology; Keratin 14 (905304) and Keratin 5 (905504) from BioLegend; Peroxidase goat anti-rabbit (PI-1000), horse anti-goat (PI-9500), and horse anti-mouse IgG (PI-2000) antibodies from Vector Labs. Keratinocyte SFM and supplements, RPM1, DMEM, DMEM F12 (1:1), fetal bovine serum (FBS) were purchased from Corning; tet system approved FBS from Clontech. Silencer select siRNA sequences against human GLI2, GLI1 and KRAS were purchased from Ambion.

Adams C.R., Htwe H.H., Marsh T., Wang A.L., Montoya M.L., Subbaraj L., Tward A.D., Bardeesy N, & Perera R.M. (2019). Transcriptional control of subtype switching ensures adaptation and growth of pancreatic cancer. eLife, 8, e45313.

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Alisertib-Mediated Signaling Pathway Study

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Alisertib was prepared and stored according to the manufacturer’s instructions (Millennium Pharmaceuticals, Inc., Cambridge, MA). Specific antibodies against p-MTOR(Ser2448), p-MEK1/2(Ser217/221), p-AKT(Ser473), p-AURKA(T288), mTOR, MEK, AKT, AURKA, RPS6KB1, p-RPS6KB1(T389), Phospho-(Ser/Thr) Phe, and β-Actin were purchased from Cell Signaling Technology (Beverly, MA). Recombinant human AURKA and RPS6KB1 proteins were obtained from Cell Sciences (Canton, MA). Specific antibodies against KRAS were purchased from Santa Cruz Biotechnology (Dallas, TX). KRAS-G12D lentiviral vector was purchased from Applied Biological Materials (Richmond, BC, Canada). Tet-One™ Inducible Expression System and Tet System Approved FBS were purchased from Clontech (Palo Alto, CA). Transfection reagent LipoJet was purchased from SignaGen Laboratories (Gaithersburg, MD). Tet-on expression system, doxycycline-free fetal bovine serum, and human cell cycle arrays were purchased from Clontech (Palo Alto, CA).

Wang-Bishop L., Chen Z., Gomaa A., Lockhart A.C., Salaria S., Wang J., Lewis K.B., Ecsedy J., Washington K., Beauchamp R.D, & El-Rifai W. (2018). Inhibition of AURKA Reduces Proliferation and Survival of Gastrointestinal Cancer Cells With Activated KRAS by Preventing Activation of RPS6KB1. Gastroenterology, 156(3), 662-675.e7.

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KSHV Infection of HUVEC Cells

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hTERT-HUVEC and primary HUVEC were grown in EBM-2 (Lonza) with the EBM-2 bullet supplement (Lonza) as previously described (56 (link), 74 (link)). TREx BCBL1 and TREx BCBL1-RTA PEL cells (57 (link)) were kindly provided by Jae Jung’s lab and grown in RPMI 1640 medium (Corning) containing Tet System Approved FBS (Clontech) and 20 µM hygromycin B (Roche). All media were supplemented with 10% heat-inactivated fetal bovine serum, 1% penicillin-streptomycin, and 1% l-glutamine. hTERT-HUVEC stably expressing the EV or FLAG-tagged vIL-6 were described previously (25 (link)). TREx-BCBL1 cells were reactivated by supplementing medium with 1 µg/ml doxycycline for 24 to 30 h. The production of purified KSHV particles (75 (link)) and KSHV-infected HUVEC (56 (link)) was described previously (also see Text S1 in the supplemental material).

Giffin L., West J.A, & Damania B. (2015). Kaposi’s Sarcoma-Associated Herpesvirus Interleukin-6 Modulates Endothelial Cell Movement by Upregulating Cellular Genes Involved in Migration. mBio, 6(6), e01499-15.

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Development of GFP-IBD Reporter Cell Lines

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The MCF7 Tet-On Advanced cell line was obtained from Clontech. The generation and characterization of the MCF7^GFP-IBD cell line has been described (24 (link)) and was used with further authentication by IDEXX BioResearch within the last 6 months. Panc 02^GFP-IBD, U-87 MG^GFP-IBD, and hTERT-HME1^GFP-IBD cell lines were developed similarly from parent cell lines purchased from American Type Culture Collection (ATCC). Briefly, GFP-IBD cloned into the pLVX-Tight-Puro vector was transfected along with pLVX-Tet-On Advanced vector (Clontech) into each cell line. Following G418 and puromycin selection, cells were induced with 1 μg/mL doxycycline and sorted to establish the IRIF reporter cell lines. The Panc 02^GFP-IBD and U-87 MG^GFP-IBD cell lines were maintained in RPMI (Invitrogen), supplemented with 10% Tet system approved FBS (Clontech). The hTERT-HME1^GFP-IBD cell line was maintained in MEBM media supplemented with MEGM SingleQuot (Lonza). For studies requiring glucose and glutamine limitation, media was prepared using DMEM base, D-glucose (Sigma), and L-glutamine solutions (Gemini Bioproducts) at appropriate concentrations with 10% FBS.

Efimova E.V., Takahashi S., Shamsi N.A., Wu D., Labay E., Ulanovskaya O.A., Weichselbaum R.R., Kozmin S.A, & Kron S.J. (2015). Linking Cancer Metabolism to DNA Repair and Accelerated Senescence. Molecular cancer research : MCR, 14(2), 173-184.

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Cell Line Culture Protocols

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HeLa epithelial cells, human kidney HEK293 cell line, neuroblastoma NB-A, and normal human fibroblasts were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). The HAP-1 wild-type (WT) and CRISPR ZFP36-knockout haploid fibroblast –like cell line were obtained from Horizon Discovery (UK) and cultured in Iscove’s Modified Dulbecco’s Medium with 10% fetal bovine serum (FBS) and 1% antibiotics. HeLa and HEK cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM), while neutroblastoma and fibroblasts were maintained in MEM (ThermoFisher, Carlsbad, CA, USA) supplemented with 10% (FBS) and antibiotics. Tet-On and Tet-Off Advanced HeLa cells were obtained from ClonTech and were maintained in DMEM supplemented with 10% Tet-System Approved FBS (ClonTech) and antibiotics and maintained in selection medium (100 μg/mL G418; Sigma).

Patel N., Khan A.O., Al-Saif M., Moghrabi W.N., AlMaarik B.M., Ibrahim N., Abdulwahab F., Hashem M., Alshidi T., Alobeid E., Alomar R.A., Al-Harbi S., Abouelhoda M., Khabar K.S, & Alkuraya F.S. (2017). A novel mechanism for variable phenotypic expressivity in Mendelian diseases uncovered by an AU-rich element (ARE)-creating mutation. Genome Biology, 18, 144.

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Inducible p21 Tet-ON Cell Lines

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Inducible p21^WAF1/Cip1 Tet-ON cell lines—Saos2- p21^WAF1/Cip1 Tet-ON and Li Fraumeni- p21^WAF1/Cip1 Tet-ON—were maintained in High Glucose DMEM (Biosera) supplemented with 10 % Tet System Approved FBS (Clontech) and 100 μg/ml penicillin and streptomycin (Invitrogen) and incubated at 37 °C and 5 % CO₂. Induction of p21^WAF1/Cip1 was conducted by treatment of the cell culture with 1 μg/ml doxocycline (Applichem) [9 (link)].

Galanos P., Pappas G., Polyzos A., Kotsinas A., Svolaki I., Giakoumakis N.N., Glytsou C., Pateras I.S., Swain U., Souliotis V.L., Georgakilas A.G., Geacintov N., Scorrano L., Lukas C., Lukas J., Livneh Z., Lygerou Z., Chowdhury D., Sørensen C.S., Bartek J, & Gorgoulis V.G. (2018). Mutational signatures reveal the role of RAD52 in p53-independent p21-driven genomic instability. Genome Biology, 19, 37.

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Culture of Human Cell Lines

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Human cervical cancer cell-derived HeLa cells, kindly supplied from Hiroyuki Sasaki at Kyushu University, Japan, were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% inactivated fetal bovine serum (FBS) at 37°C with 5% CO₂. Human embryonic kidney cell-derived Flp-In T-REX 293 cells (Life Technologies) were cultured in DMEM containing 10% heat-inactivated Tet-system approved FBS (Clontech) without tetracycline and 10 ng/ml of Hygromycin B at 37°C with 5% CO₂.

Komori C., Takahashi T., Nakano Y, & Ui-Tei K. (2020). TRBP–Dicer interaction may enhance HIV-1 TAR RNA translation via TAR RNA processing, repressing host-cell apoptosis. Biology Open, 9(2), bio050435.

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