A mixture of α7-containing and NACHO-containing lentiviral particles (10:1) was used to transduce HEK T-Rex 293 cells (Thermo Fisher). The resulting inducible cell line was cultured in suspension flasks in Freestyle 293 expression medium supplemented with blasticidin (5–10 µg/ml final) and FBS (1%) in an orbital incubator at 37 °C and 8% CO2. Protein expression was induced at a density of 3–4 × 106 cells/ml by the addition of doxycycline (10 µg/L final). The cells were further cultured for 48 h before harvesting by centrifugation.
Hek 293 t rex cells
Manufactured by Thermo Fisher Scientific
Sourced in Denmark
HEK 293 T-REx cells are a human embryonic kidney cell line that can be used for a variety of cell-based applications. These cells are genetically modified to allow for inducible gene expression using the tetracycline regulatory system.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Pog44 flp recombinase expression vector, by Thermo Fisher Scientific (2 mentions)
Transit transfection reagent, by Mirus Bio (1 mentions)
T rex hek293 cells, by Thermo Fisher Scientific (1 mentions)
Zeocin, by Thermo Fisher Scientific (1 mentions)
Hek293t, by American Type Culture Collection (1 mentions)
8 protocols using hek 293 t rex cells
1
Inducible Lentiviral Protein Expression
2
Stable Inducible HEK T-REx 5-HT3A Expression
3
Tetracycline-Inducible Stable Cell Line Generation
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HEK 293 T-REx cells (Life Technologies) were maintained in DMEM and 10% fetal bovine serum supplemented with 2 mM glutamine, and maintained in a humidified atmosphere of 5% CO2 at 37°C. Tetracycline-inducible stable cell lines were generated by electroporation and maintained in media supplemented with selective compounds zeocin or blasticidin and hygromycin. Positive clones were screened by co-immunoprecipitation and western blotting. Induction was induced by 1 μg/ml tetracycline for 16 h. Cells were synchronized by serum starvation (0% FBS) for 48 h before use. Transient transfection of HEK 293 T-REx cells with the pCAG plasmid was achieved with TransIT transfection reagent (Mirus Bio).
4
Conditional CCR5 and ACKR2 Expression in HEK293 Cells
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Conditional expression of CCR5 and ACKR2 was achieved using HEK293 T-Rex cells (Life Technologies), which stably express the tetracycline-responsive repressor protein and inhibit gene expression downstream to tetracycline-responsive operon. HEK293 T-Rex cells were maintained in complete D-MEM with 25 μg/ml blasticidin and were transfected using lipofection (Lipofectamine 2000, Invitrogen) with pcDNA4/Tet-on plasmids encoding HA-tagged ACKR2 and CCR5 under control of a tetracycline-responsive promoter. Cells were selected using 100 μg/ml zeocin (Life Technologies) and receptor expression was induced incubating cells with 1 μg/ml tetracycline. Analysis was performed 24 h after receptor expression induction.
5
Cell Line Maintenance Protocols
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BSC-1 (ATCC CCL-26) and HEK293T (ATCC CRL-11268) cells were maintained in DMEM containing 10% fetal bovine serum (FBS) and penicillin-streptomycin (50 μg/ml; PS). RK-13 (ATCC CCL-37) cells were maintained in minimum essential medium (MEM), supplemented as above. HeLa (ATCC CCL-2) cells were cultured in MEM, supplemented above with the addition of 1% nonessential amino acids. MEFs (Xrcc5+/+/Tp53−/−, and Prkdc−/−) were grown in DMEM containing 15% FBS and PS. HEK293 TRex cells (Life Technologies) were grown in DMEM supplemented with 15% (v/v) FBS, PS, blasticidin (50 μ g/ml), and zeocin (100 μ g/ml).
6
Generation of CXCR4b Mutant Cell Line
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The plasmid pCDNA5/FRT/TO-cxcr4bI7E,I8E-Kate2-IRES-GFPF was generated from the plasmid pCDNA5/FRT/TO-cxcr4b-Kate2-IRES-GFPF (see above) using Gibson cloning. The primer 5’-GGTGGAATTCATGGAATTTTACGATAGCGAAGAA TTAGACAACAGCTCTGACTCC-3’ was used to mutate the codons for isoleucines 7 and 8 to codons for glutamates. 0.4 μg of the plasmid and 3.6 μg of pOG44 Flp-recombinase expression vector (Thermo Fisher Scientific, catalog number V600520) were co-transfected into a 30 % confluent 6-well plate of T-REx HEK 293 cells (Thermo Fisher Scientific, catalog number A15008) using Lipofectamine 2000 (Thermo Fisher Scientific, catalog number 11668027). The cell line was re-plated onto 10 cm plates in DMEM + 10% FBS + 100 μg/mL hygromycin + 15 μg/mL blasticidin and maintained in selection media for 10 days to select for clones with stably integrated constructs. Colonies that emerged after 10 days were transferred into 96-well plates using cloning cylinders and expanded. Early passage clones were frozen. Clones were analyzed for expression of Kate2 and GFPF by live confocal microscopy following doxycycline induction, and tested for sensitivity to 1mg/mL zeocin.
7
Generation of CXCR4b Mutant Cell Line
8
High-throughput Kv7 Channel Recordings
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High-throughput patch-clamp recordings were obtained similar to previous descriptions using the Qube 384 automated voltage-clamp platform (Sophion Bioscience A/S, Copenhagen, Denmark),39–41 (link) and adherent T-REx HEK293 cells (ThermoFisher), stably transfected with an expression vector containing the full-length cDNA coding for the human Kv7.2, Kv7.3, or Kv7.5 α-subunits (in Kv7.2/Kv7.3 or Kv7.3/Kv7.5 cell lines). The human Kv7.2, Kv7.3, and Kv7.5 constructs used correspond to GenBank accession NM_172 107 , NM_004 519 , and NM_019842NM , respectively. Multihole plates were used and each recording represents the sum of currents (10 sites per well), filtered for minimum seal resistance (>30 MΩ membrane resistance) and minimum current signal size (>2 nA) to minimize contamination of the signal with endogenous currents. Leak subtraction protocols and compensation were not applied.
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