Human embryonic kidney cells HEK293T/17 (ATTC CRL‐11268) were cultured in DMEM (Sigma; D6546) supplemented with 10% (v/v) heat‐inactivated foetal bovine serum (Gibco, Gaithersburg, MD, USA), 2 mm l ‐glutamine and 100 units/ml of penicillin‐streptomycin. Cells were incubated at 37 °C in a humidified incubator with 5% (v/v) CO2. For luciferase assays, 3 × 105 cells/well were seeded in 12‐well tissue culture plates in the absence of antibiotics. The next day, plasmids (0.5 μg of pGL3‐Avp promoter and 0.5 μg of pcDNA3 or pcDNA3‐Creb3l1 and 0.05 μg of pRL‐TK vector/well) were transfected with FuGENE HD transfection reagent (Promega). The culture media was replaced with fresh media at 8 h after transfection. Luciferase assays were performed using Promega's Dual‐Luciferase® Reporter Assay kit. At 36 h after transfection, culture media was removed and cells were washed with phosphate‐buffered saline and lysed with 500 μl of the supplied lysis buffer. Luciferase activity was measured using a Lumat LB 9507 Luminometer (Berthold Technologies GmbH, Bad Wildbad, Germany).
Hek293t 17
Manufactured by Merck Group
Sourced in United States
The HEK293T/17 is a cell line derived from human embryonic kidney cells. This cell line is commonly used in research and laboratory applications that require a robust and reliable cell model.
Automatically generated - may contain errors
Lab products found in correlation
Hek293t 17, by American Type Culture Collection (3 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (2 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Lumat lb 9507 luminometer, by Berthold Technologies (1 mentions)
Dual luciferase reporter assay kit, by Promega (1 mentions)
Fugene hd transfection reagent, by Promega (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Sh sy5y, by Merck Group (1 mentions)
Sk n be 2 c, by Merck Group (1 mentions)
Minimum essential medium (mem), by Merck Group (1 mentions)
Imr 32, by Merck Group (1 mentions)
Sk n as, by Merck Group (1 mentions)
Dmem f12 medium, by Merck Group (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Sh sy5y, by American Type Culture Collection (1 mentions)
Imr 32, by American Type Culture Collection (1 mentions)
Sk n as, by American Type Culture Collection (1 mentions)
Sk n be 2 c, by American Type Culture Collection (1 mentions)
5 protocols using hek293t 17
1
Luciferase Assay for Creb3l1 Activity
2
Cell Culture Protocols for Neuroblastoma and HEK293T Lines
3
Transfecting HEK293T/17 Cells with GRM3 Constructs
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The human embryonic kidney cell line HEK293T/17 (ATCC CRL-11268) was maintained in Dulbbeco's modified Eagle's medium (DMEM; Sigma D6545) containing 4.5 g/L glucose and supplemented with 10% fetal bovine serum (FBS) (Sigma F9665) and 4 mM l -glutamine (Sigma G7513). Cells were seeded at a density of 5 × 104 cells/cm2 in Chamber Slides (Nunc™ Lab-Tek™ II Chamber Slide™ System) for immunocytochemistry or T-75 flasks for western blots. Cells were grown for 24 h and then transfected with a PCI neo construct containing the open reading frame for either full length human GRM3 or GRM3Δ4 using a standard polyethylenimine (Aldrich 40,872-7 average MW 25000) protocol. Briefly, human GRM3 construct (533.33 ng/μL; 200 ng/cm2) was mixed with 20% glucose at a ratio of 1:3 glucose to DNA, PEI (5.6 mg/mL) was then added to the mix at a ratio of 1:3.3 (PEI to DNA glucose) and incubated for 5 min at room temperature. The mixture was added to fresh culture media (DMEM 4.5 g/L glucose, 10% FBS and 2 mM l -glutamine) and cells were incubated for 24 h, following which, the media was exchanged. Cells were incubated for a further 24 h before harvesting.
4
Cultivation of Human Cell Lines
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MDA-MB-453, T-47D, 22Rv1, VCaP, ZR75–1, MDA-MB-231, MCF7 and HEK293T/17 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). MFM-223, Cal-51 and Cal-148 cells were obtained from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). LNCaP-95 cells were a kind gift from Dr. Alan K. Meeker (Johns Hopkins University). MDA-MB-453, T-47D, ZR75–1 and 22Rv1 cells were maintained in RPMI-1640 medium containing 10% fetal bovine serum (FBS); VCaP cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% FBS, 1% sodium pyruvate, 1% MEM non-essential amino acids and 0.1 nM 5α-dihydrotestosterone (DHT; Sigma, St. Louis, MO, USA); MDA-MB-231 cells were maintained in RPMI-1640 medium containing 5% FBS; MCF7 cells were maintained in Eagle's Minimum Essential Medium (EMEM) containing 10% FBS, 2 mM L-glutamine, sodium pyruvate and 0.2 U/mL insulin; MFM-223 cells were maintained in EMEM containing 10% FBS, 2 mM L-glutamine, and insulin-transferrin-sodium selenite (ITS) supplement; Cal-51 cells were maintained in DMEM containing 10% FBS; Cal-148 cells were maintained in DMEM containing 10%FBS, 2 mM L-Glutamine and EGF (1ug/100mL); HEK293T/17 cells were maintained in DMEM containing 10% FBS and 20 mM HEPES.
5
Lentiviral shRNA Knockdown Assay
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shRNA vectors (Supplementary Table 6 ) or SHC002 MISSION Non-Target shRNA control vector (Sigma) along with lentivirus packaging plasmids pCMV-dR8.2 and pCMV.VSV.G (Addgene) were transfected in HEK293T/17 (ATCC #CRL: 11268). Target LNCaP, PC3, or RWPE-1 cells (ATCC #: CRL-11609) grown in Keratinocyte-SFM 1X (Gibco) were seeded 24 hours following transfection. Supernatants from HEK293T/17 cells were collected 48 hours after transfection, passed through a 0.45-μm SFCA filter, and applied on target cells along with Polybrene (8 μg/mL) (Sigma). Cells were selected with puromycin (Wisent) for 72 hours. Immediately following selection, cells were plated for growth assays and counted at the times indicated.
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