Dulbecco's modified Eagle's medium (DMEM) and DMEM-F12 containing 10% heat-inactivated fetal calf serum (FCS) (Thermo Fisher) were used to culture human HEK293T (ATCC), HeLa cells (ATCC), Flp-In-293 (Thermo Fisher), and murine IMCD3 (ATCC) cells, respectively. Plasmids were delivered to the cells by using Plus Reagent and Lipofectamine LTX (Invitrogen) following the manufacturer's protocol. HEK293T cells stably expressing FLAG/HA-tagged human PRPF4 was generated using the pcDNA5/FRT-PRPF4 plasmid and Flp-In-293 cells according to the manufacturer's protocol (Thermo Fisher). Briefly, Flp-In-293 cells were transfected with a 9:1 ratio of pOG44:pcDNA5/FRT-PRPF4 plasmids using Lipofectamine 2000 (Invitrogen) and 48 h post-transfection, cells were selected in medium containing hygromycin. Stable clones resistant to hygromycin were expanded and tested for stable expression of FLAG-PRPF4 by Western blotting.
Flp in 293
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Flp-In-293 system is a tool for the creation of stable mammalian cell lines. It facilitates the integration of a gene of interest into a specific chromosomal location in human embryonic kidney (HEK) 293 cells. The system provides a consistent and reliable method for generating stable cell lines expressing the desired protein.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions)
Hek293, by American Type Culture Collection (3 mentions)
Hek293t, by American Type Culture Collection (3 mentions)
Fetal bovine serum (fbs), by Merck Group (3 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Nacalai Tesque (2 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (2 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
X tremegene 9, by Roche (1 mentions)
X tremegene hp, by Roche (1 mentions)
Heat inactivated fetal calf serum, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Dulbecco s modified eagle s medium, by Merck Group (1 mentions)
Flp in system, by Thermo Fisher Scientific (1 mentions)
Sirna id s4219, by Thermo Fisher Scientific (1 mentions)
17 protocols using flp in 293
1
Establishing stable cell lines expressing PRPF4
2
RMCE-in Cell Line Generation
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RMCE-in HEK293 or HeLa and Flp-in™-293 (Life Technologies #R750-07) were seeded at 60% confluence in a 12-well plate and all transfections were made in triplicates. Cells were transfected with 250 ng donor and PGK-FlpO plasmid (5067 bp) or 250 ng empty pUC 19 control plasmid using X-tremeGENE 9 in HEK293 or XtremeGENE HP (Roche) in HeLa cells. All cells were transferred using 0.05% Trypsin-EDTA (Gibco) to 60 cm2 petri dishes the following day. Day three, cells were subject to HygR selection (100 μg/ml for HEK293 cells and 250 μg /ml for HeLa cells) and continuously HygR renewed every 3-4th day until harvested. Six million murine RMCE-in ES cells were electroporated using 10 ug donor and 10 ug FlpO plasmid (5067 bp) or 10 ug empty pUC 19 control plasmid. ES cells were seeded on mitotically inactivated murine fibroblasts and selected for 3 days with HygR (125 μg/ml).
3
HEK 293 Cells siRNA Knockdown
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HEK 293 cells (Flp-In-293, from Life Technologies) were grown in Dulbecco's modified Eagle's medium (DMEM; from Sigma) supplemented with 2 mM L-glutamine (Gibco) and 10% heat-inactivated fetal calf serum (Gibco). Transfections of siRNA were carried out using Lipofectamine RNAiMAX (Life Technologies) following the manufacturer's protocol. The following siRNAs were used: negative-control from Microsynth (sense strand AGGUAGUGUAUCGCCUUGTT) and si-HNRNPC1/2 (sc-35577 from Santa Cruz Biotechnologies), both applied at 20 nM in 2.5 mL DMEM on six-well plates.
4
Generating Ouabain-Resistant ATP1A3 Mutants
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For the Flp-In system in 293 cells, a DNA construct containing 3.5 kb of ATP1A3 full-length cDNA (GenBank accession NM_152296.4 ) was purchased from OriGene. 3042 bp of ATP1A3 ORF fragment was PCR-amplified using primers 5′-aaaAAGCTTggccacc-ATGGGGGACAAGAAAGATG (forward) and 5′-aaaGGATCCTCAGTAGTAGGTTTCCTTC (reverse) and subcloned in pcDNA5/FRT/TO vector using HindIII and BamHI restriction sites to produce pcDNA5/FRT/TO/ATP1A3-OR (ouabain resistant) for Tet-inducible expression in Flp-in 293 cell lines (ThermoFisher Scientific). Site-directed mutagenesis by PCR was performed to generate mutations including D366H, R463C, S729Y, D742Y, D743H, R756C, R756H, R756L, E815K, E818K, D923N, and L924P using pcDNA5/FRT/TO/ATP1A3-OR. The subcloning and site-directed mutagenesis were performed by Mutagenex (now Molecular Biology Core, The Ohio State University, Department of Surgery). All constructs were confirmed by Sanger sequencing for whole ORF sequences. For COS-1 cells, plasmids were mutagenized by PCR using the quick-change mutagenesis kit (11 (link), 47 ).
5
HEK293 and Flp-In-293 Cell Culture
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HEK293 (human [Homo sapiens] embryonic kidney) cells were obtained from ATCC [https://www.atcc.org/ ] and cultured in Dulbecco´s Modified Eagle Medium (DMEM, low glucose, GlutaMaxTM Supplement, pyruvate) supplemented with 10% fetal bovine serum, 100 I.U. ml−1 penicillin, 100 µg ml−1 streptomycin, and 1.0 mg ml−1 G418. Flp-In™-293 (human [Homo sapiens] embryonic kidney) cells were obtained from Thermo Fisher Scientific and stable cell lines were generated using the Flp-In™ system from Invitrogen (Thermo Fisher Scientific, USA). Flp-In™-293 cells were cultured in DMEM (high glucose, GlutaMaxTM Supplement, pyruvate) supplemented with 10% fetal bovine serum, 100 I.U. ml−1 penicillin, 100 µg ml−1 streptomycin, and 100 µg ml−1 hygromycin B. All cell lines were incubated at 37 °C with 10% CO2.
6
DART System with TRDMT1 Knockout U2OS Cells
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U2OS TRE40 (link), TRDMT1 KO U2OS TRE22 (link), Flp-in 293 (Thermo, Cat#R75007), HS578T and MDA-MB-231 cells were cultured in Dulbecco’s modified Eagle medium (DMEM, Cat#12–604 F, Lonza; Basel, Switzerland) with 10% (vol/vol) FBS at 37 °C with 5% CO2. The U2OS-TRE cells used for the DART system have been described in previous articles40 (link). The HS578T and MDA-MB-231 cells are gifts from Dr. Leif W. Ellisen. For plasmid and siRNA transfection, Lipofectamine 2000 and Lipofectamine RNAiMax (Invitrogen; Carlsbad, CA, USA) were used following the manufacturer’s standard protocol, respectively. The siRNA for TRDMT1 was purchased from Invitrogen (siRNA ID: s4219, Cat#: 4392420). Other siRNAs include siBRCA1 (L-003461-00, Dharmacon), and siBRCA2 (GS675, Qiagen).
7
Mesenchymal Stem Cell Cultivation and Stimulation
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Primary mouse C57BL/6J BM-derived MSCs were purchased from Cyagen Biosciences. 293FT, Flp-In-293, and Flp-In-Chinese hamster ovary cells were purchased from Thermo Fisher Scientific. The C3H10T1/2 cell line was purchased from the American Type Culture Collection. All of the cells were cultured in DMEM (Nacalai Tesque, Japan) supplemented with 10% fetal bovine serum. Lipofectamine 2000 (Thermo Fisher Scientific) was used for gene transfection into 293FT cells. Elastically supported surface dishes that have polydimethylsiloxane layers adjusted to a stiffness of 1.5 or 28 kPa were purchased from ibidi (µ-Dish, 35 mm, high elastically supported surface). Before seeding the cells, the surfaces of the dishes were coated with type I collagen (Col1a1; Millipore). For experiments under hypoxic conditions, cells were cultured in a humidified CO 2 /multigas incubator (APM-30D, ASTEC, Japan) with 1% O 2 , 5% CO 2, and 94% N 2 at 37°C. We used 20 and 0.25 ng/mL TGF-β to stimulate C3H10T1/2 and Flp-In Chinese hamster ovary cells, respectively.
8
Generating stable Flp-In 293 cell lines
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Flp-In 293 cells (Invitrogen) were cultured in Dulbecco's modified Eagle Medium (DMEM) (Nacalai Tesque, Kyoto, Japan) containing 10% fetal bovine serum (FBS) (Biowest, Nuaillé, France), 100 units/ml penicillin and 100 μg/ml streptomycin (Nacalai Tesque) and maintained in a 5% CO2 incubator at 37°C. Flp-In 293 cells were co-transfected with pcDNA5/FRT-FLAG-NLS-DMD-Exon57_58_59(short-Intron57)-DsRed-EGFP and pOG44 (the flp recombinase expression plasmid) (Invitrogen). Stable cell lines were selected by 50 μg/ml hygromycin B (Invitrogen).
9
Bacterial Hosts and HEK Cell Transfection
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The bacterial hosts used were E. coli K12 strains: TAP114 (lacZ) deltaM15 (46 (link)) and S17–1 lambda pir (47 (link)). Escherichia coli cells were grown and plated on Luria-Bertani rich medium with the appropriate antibiotics. Plasmid transformations were performed by electroporation (48 ).
Human embryonic kidney cells HEK293 (ATCC® CRL1573™), HEK293T (ATCC® CRL-3216™), and Flp-In™-293 (Invitrogen) were cultured in Dulbecco's modified Eagle's medium (DMEM). For transient transfections HEK293T cells (∼6 × 105) were plated in a six-well plate and 24 h later treated with 3 μg of the proper plasmid DNA using PureFection Transfection Reagent (System Biosciences, Mountain View, CA, USA). For the human native ‘attB’ sites chromosomal assay transfection, Flp-In™-293 cells (∼6 × 105) were plated in a six-well plate and 24 h later treated with 5.5 μg of the proper plasmid DNA using Mirus Transfection Reagent (Mirus, WI, USA). For the CTNS and DMD chromosomal assay transfection, HEK293 cells (∼6 × 105) were plated in a six-well plate and 24 h later treated with 3 μg of the proper plasmid DNA using PureFection Transfection Reagent.
Human embryonic kidney cells HEK293 (ATCC® CRL1573™), HEK293T (ATCC® CRL-3216™), and Flp-In™-293 (Invitrogen) were cultured in Dulbecco's modified Eagle's medium (DMEM). For transient transfections HEK293T cells (∼6 × 105) were plated in a six-well plate and 24 h later treated with 3 μg of the proper plasmid DNA using PureFection Transfection Reagent (System Biosciences, Mountain View, CA, USA). For the human native ‘attB’ sites chromosomal assay transfection, Flp-In™-293 cells (∼6 × 105) were plated in a six-well plate and 24 h later treated with 5.5 μg of the proper plasmid DNA using Mirus Transfection Reagent (Mirus, WI, USA). For the CTNS and DMD chromosomal assay transfection, HEK293 cells (∼6 × 105) were plated in a six-well plate and 24 h later treated with 3 μg of the proper plasmid DNA using PureFection Transfection Reagent.
10
HEK293T Cell Culture and Genetic Manipulation
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