HeLa S3 cells (RIKEN BioResource Research Center) were maintained in DMEM, high glucose (Wako Chemicals), supplemented with 10% FBS. HEK293 T-REx (Thermo Fisher Scientific) cells were maintained in DMEM, high glucose, GlutaMAX Supplement (Thermo Fisher Scientific) and supplemented with 10% FBS. All the cells were maintained in a humidified incubator at 5% CO2 and 37°C. The stable cell lines of HEK293 T-REx were selected following the manufacturer’s instructions. The sex of the HeLa S3 and HEK293 cells is female.
Hek 293 trex
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The HEK 293 Trex is a cell line commonly used in biological research. It is a derivative of the HEK 293 cell line, which is a commonly used human embryonic kidney cell line. The HEK 293 Trex cell line is designed for tetracycline-regulated gene expression.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Corning (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Turbofect, by Thermo Fisher Scientific (2 mentions)
High glucose, by Thermo Fisher Scientific (1 mentions)
Dmem high glucose, by Fujifilm (1 mentions)
Hela s3 cells, by RIKEN BioResource Center (1 mentions)
Glutamax supplement, by Thermo Fisher Scientific (1 mentions)
Hela s3, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Corning (1 mentions)
Complete dmem, by Corning (1 mentions)
Hek293t, by American Type Culture Collection (1 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Puromycin, by Santa Cruz Biotechnology (1 mentions)
Dulbecco modified eagle medium (dmem), by GE Healthcare (1 mentions)
Zeocin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Wisent (1 mentions)
Penicillin, by Wisent (1 mentions)
Streptomycin, by Wisent (1 mentions)
Dulbecco modified eagle medium (dmem), by Wisent (1 mentions)
9 protocols using hek 293 trex
1
Maintenance of HeLa S3 and HEK293 T-REx Cell Lines
2
Culturing HeLa S3 and HEK293 T-REx Cells
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HeLa S3 and HEK293 T-REx (Thermo Fisher Scientific) cells were maintained in DMEM supplemented with 10% FBS in a humidified incubator at 5% CO2 and 37°C. The stable cell lines of HEK293 T-REx were selected following the manufacturer's instructions.
3
Investigating PDI Proteins in ATF6 Pathway
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HEK293T-Rex (Invitrogen) cells were cultured in complete DMEM (CellGro) supplemented with 10% FBS (CellGro) and penicillin/streptomycin (CellGro). Lentiviruses encoding a mix of shRNAs that are either non-silencing or directed against PDIA1, PDIA3, PDIA4, PDIA5, or PDIA6 were transduced into HEK293T-Rex with 1–5 mL of virus in media containing 5 mg/mL polybrene (see Supplementary File 1 for lentivirus production methods). Stable cell lines were selected by culturing in puromycin (5 μg/mL) before characterization. The expression of the ATF6 target gene BiP in cells treated with 147 (10 μM) or thapsigargin (0.5 μM) was then measured by qPCR as described previously.
4
Cell Line Culture Protocols
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BSC-1 (ATCC CCL-26), CV-1 (ATCC CCL70), HEK 293T (ATCC CRL-11268) and A549 (ATCC CCL-185) cells were maintained in Dulbecco’s modified minimal essential medium (DMEM) containing 10% fetal bovine serum (FBS) and penicillin/streptomycin (50 μg/ml). RK-13 (ATCC CCL-37) and TK-143 (ATCC CRL-8303) cells were grown in minimum essential medium (MEM) and supplemented as above. HeLa (ATCC CCL-2) cells were grown in MEM with addition of non-essential amino acids (1%) and supplemented as above. HEK 293 Trex (Invitrogen) cells were maintained in DMEM containing 10 μg/ml blasticidin, 100 μg/ml zeocin and supplemented as above.
5
Establishment of HEK 293 Trex Clones Expressing Protein 169
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HEK 293 Trex (Invitrogen) empty cells were transfected with 169 (pcDNA4 TO) using TransIT-LT1. Transfected cells were selected in the presence of zeocin and were serially diluted to obtain individual clones. Expression of protein 169 within these clones were analyzed by immunoblotting and immunofluorescence. In the chosen clone at least 90% of cells were expressing protein 169.
6
Stable Cell Line Generation
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HeLa, HEK293 T-REx (Invitrogen) as well as SW480 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM/GE Healthcare) including 10% foetal bovine serum (Thermo Scientific) and 1% penicillin/streptomycin (Thermo Scientific) in a humidified environment with 5% CO2. Stable cell lines were generated using the piggybac transposon system [32 (link)]. Positive clones were picked after puromycin selection (Santa Cruz 1 µg/mL). Quantification of gels and Western blots was performed with ImageJ. P values were calculated by the Student’s t test. Statistical significance: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.
7
Cell Culture Protocol for HEK293 and MDCKII
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Cell lines used in this study were HEK293T, HEK293 T-REx (Invitrogen), and MDCKII (gift from Dr. David Bryant, Beatson Institute, Glasgow, UK). HEK293 cells were grown in DMEM (Wisent) with 10% FBS (Wisent) and 100 U/ml penicillin and 100 µg/ml streptomycin (Wisent). MDCKII were grown in DMEM with 5% FBS. HEK293 T-REx cells were additionally maintained with 5 µg/ml blasticidin (BioShop) and 100 µg/ml zeocin (Invitrogen). Cells were cultured at 37°C in a humidified incubator with 5% CO2.
8
Generating Stable HEK293T-REx Cell Lines
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HEK293T-REx (Invitrogen)
cells were cultured
in DMEM (CellGro) supplemented with 10% fetal bovine serum (CellGro)
and 1% penicillin/streptomycin/glutamine (CellGro). HEK293T-REx cells
were cultured in 5 mg/mL blasticidin (InvivoGen) to maintain the tet
repressor. HEK293T-REx cells were transiently transfected using calcium
phosphate. Stable, clonal HEK293T-REx cell lines were selected from
transiently transfected populations using 500 μg/mL G-418 sulfate
(Cellgro) for cells expressing pcDNA-DEST40 or pTREx-DEST30 vectors.
All stable cell lines were maintained in the appropriate selective
antibiotics.
cells were cultured
in DMEM (CellGro) supplemented with 10% fetal bovine serum (CellGro)
and 1% penicillin/streptomycin/glutamine (CellGro). HEK293T-REx cells
were cultured in 5 mg/mL blasticidin (InvivoGen) to maintain the tet
repressor. HEK293T-REx cells were transiently transfected using calcium
phosphate. Stable, clonal HEK293T-REx cell lines were selected from
transiently transfected populations using 500 μg/mL G-418 sulfate
(Cellgro) for cells expressing pcDNA-DEST40 or pTREx-DEST30 vectors.
All stable cell lines were maintained in the appropriate selective
antibiotics.
9
Establishment of Inducible Cell Lines
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HEK293 (HEK293 T-Rex purchased from Invitrogen) and HaCat cells (purchased from Cell Line Service) were cultivated in DMEM (PAN-Biotech) supplemented with 10% (Fetal Calf Serum) FCS (PAN-Biotech) and Penicillin/Streptomycin (HyClone). Medium for MDA-MB486 (purchased from ATCC) was further supplemented with non-essential amino acids (HyClone). Cells were kept at 37 °C, 5% CO 2 in a humidified atmosphere. For transient transfection experiments 1 × 10 4 HEK293 cells per well were seeded into polyethyleneimine (PEI)-coated [39] (link) transparent 96-well plates, incubated for 24 h at 37 °C, and transfected with TurboFect (Thermo Fisher Scientific) according to the manufacturer's instructions with 10 ng reporter plasmid per well. For the generation of stable cell lines, HaCat or MDA-MB486 cells were transfected with pGVL8 constructs using the piggy bac transposon system [38] (link). After 48 h cells were selected with 1.5 µM puromycin added to the normal growth medium. Positive clones were picked after puromycin selection. Clones were tested for induction with 100 µM benzylideneacetone and selected for low basal NlucP levels and high induction rate. For more information on stable cell lines see S1 Table.
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