Cells were cultivated in DMEM (Dulbecco's modified Eagle's medium; Gibco) with 10% FBS (Gibco), 2 mM GlutaMAX (Gibco), 10 000 units/ml penicillin and 10 mg/ml Streptomycin (Invitrogen) at 37°C, 5% CO2. Stable cell lines were generated using the Flp-in T-REx293 system according to the manufacturer's instructions (Invitrogen).
Flp in t rex293 system
Manufactured by Thermo Fisher Scientific
The Flp-in T-REx293 system is a cell line used for the inducible, high-level expression of recombinant proteins in human embryonic kidney (HEK) 293 cells. It combines the Flp-In system for site-specific integration of the gene of interest with the Tet Repressor (T-REx) system for tetracycline-inducible expression.
Automatically generated - may contain errors
Lab products found in correlation
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Hepg2 cell, by Thermo Fisher Scientific (1 mentions)
Hygromycin b, by Thermo Fisher Scientific (1 mentions)
Fugene 6 transfection reagent, by Roche (1 mentions)
4 protocols using flp in t rex293 system
1
Establishing Flp-in T-REx293 Cell Lines
2
Stable Inducible WT and D2594K IP3R1 HEK293 Cell Lines
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Briefly, as previously described [46 (link)], stable inducible WT- and D2594K-IP3R1 expressing HEK293 cell lines were generated using full-length ITPR1 cDNA that was sub-cloned into an inducible expression vector, pcDNA5/FRT/TO. The recombinant expression of rat IP3R1 was achieved by the Flp-In T-REx-293 system (Invitrogen). Cells were incubated at 37 °C under 5% CO2 with DMEM supplemented with 0.1 mM nonessential amino acids, 2 mM L‐glutamine, 100 units of penicillin/ml, 1% streptomycin/ml, and 5% fetal bovine serum. Cells were selected for Flp positive in 200 µg/ml hygromycin selection medium. Induction of IP3R1-WT or IP3R1-D2594K expression was initiated upon incubation with DMEM containing 1 μg/ml tetracycline.
3
Genome Editing of Nrf1α and Nrf2 in HepG2 Cells
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Nrf1α−/− cells were established by TALENs-led genome editing with HepG2 cells [26 ], whereas Nrf2−/− cells were constructed by CRISPR/Cas9-editing system with HepG2 cells [29 ]. These cell lines expressing Nrf1α and Nrf2, as well as an empty control, were established by using the Flp-In™ TREx™-293 system with HepG2 cells (Invitrogen) [30 ]. These cell lines were maintained in DMEM supplemented with 5 mM glutamine, 10% (v/v) fetal bovine serum (FBS), and 100 units/mL of either penicillin or streptomycin, in the 37 °C incubators with 5% CO2. In addition, some cell lines were transfected for 8 h with the indicated constructs mixed with the Lipofectamine®3000 agent in the Opti-MEM (gibca, Waltham, MA, USA). The cells were then allowed for recovery from transfection in a fresh complete medium for 24 h before the other experiments were conducted.
4
Inducible Overexpression of D1/H3 Chimeras
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Inducible overexpressing
cell lines for D1 and D1/H3 chimeras were generated using the Flp-In
T-REx-293 system (Invitrogen). The pcDNA5/FRT/TO-CBP-SBP-D1/H3 and
pOG44 (Invitrogen) plasmids were co-transfected using the FuGENE6
transfection reagent (Roche) into FlpIn-TReX HEK293 cells, according
to manufacturer’s instructions. Cells that successfully incorporated
CBP-SBP-D1/H3 into the genome were selected by the addition of 200
μg/mL Hygromycin b (Invitrogen) to the medium and further cultured
in tetracycline-reduced Dulbecco’s modified Eagle’s
medium with 10% fetal bovine serum, 1% glutamine, 1% penicillin, and
streptomycin. The expression of constructs in doxycycline-induced
cells was confirmed by Western blot using the anti-SBP antibodies
(Millipore).
cell lines for D1 and D1/H3 chimeras were generated using the Flp-In
T-REx-293 system (Invitrogen). The pcDNA5/FRT/TO-CBP-SBP-D1/H3 and
pOG44 (Invitrogen) plasmids were co-transfected using the FuGENE6
transfection reagent (Roche) into FlpIn-TReX HEK293 cells, according
to manufacturer’s instructions. Cells that successfully incorporated
CBP-SBP-D1/H3 into the genome were selected by the addition of 200
μg/mL Hygromycin b (Invitrogen) to the medium and further cultured
in tetracycline-reduced Dulbecco’s modified Eagle’s
medium with 10% fetal bovine serum, 1% glutamine, 1% penicillin, and
streptomycin. The expression of constructs in doxycycline-induced
cells was confirmed by Western blot using the anti-SBP antibodies
(Millipore).
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