HEK293 cells (Human embryonic kidney cells) were purchased from ATCC and grown in Earle's minimal essential medium (MEM) supplemented with 10% fetal calf serum, 200 mM GlutaMAX, 100 mM sodium pyruvate and 50 U/mL penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO, USA) under standard growth conditions at 37°C and 5% CO2. COS7 cells (African green monkey kidney cells) were purchased from ATCC and grown in Dulbecco's modified Earle's medium (DMEM) with the same supplements added for HEK293 cells and under the same growth conditions.
Hek293 cell
Manufactured by American Type Culture Collection
Sourced in United States, Germany, China, United Kingdom, Japan, Switzerland, France
HEK293 cells are a widely used cell line derived from human embryonic kidney cells. They are immortalized, adherent cells that can be cultured in vitro. HEK293 cells are commonly used in various research applications, such as protein expression, transfection studies, and viral vector production.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (178 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (146 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (109 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (104 mentions)
Streptomycin, by Thermo Fisher Scientific (65 mentions)
Penicillin, by Thermo Fisher Scientific (57 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (49 mentions)
Hek293 cell, by Thermo Fisher Scientific (45 mentions)
Glutamax, by Thermo Fisher Scientific (36 mentions)
L glutamine, by Thermo Fisher Scientific (31 mentions)
Hela cell, by American Type Culture Collection (29 mentions)
Fetal bovine serum (fbs), by Merck Group (29 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (28 mentions)
Trypsin edta, by Thermo Fisher Scientific (27 mentions)
Rpmi 1640, by Thermo Fisher Scientific (24 mentions)
Hek293t cell, by American Type Culture Collection (22 mentions)
Dual luciferase reporter assay system, by Promega (22 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (21 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (19 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (19 mentions)
1 322 protocols using hek293 cell
1
Cell Culture Protocols for HEK293 and COS7
2
Cloning and Expressing Chlopsid Fluorescent Protein in HEK293 Cells
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A pCS2+ vector was used for cloning of Chlopsid FP I for expression in HEK293 cells (ATCC, USA) using Kpn-SphI cloning sites. Plasmids were prepared by Genscript USA, Inc. The expression was driven in HEK293 cells (ATCC, USA) by CMV promotor. The HEK293 cell line was maintained in Dulbecco’s Modified Eagle Medium (High Glucose) (DMEM) (Invitrogen, USA), supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, USA), in a 37°C incubator with 5% CO2. Transient transfection was performed using 2 μg of DNA per 35 mm dish and 5μg of Lipofectamine 2000 (Invitrogen, NY). Cells were imaged on a custom-made 2-photon microscope using a Chameleon Ti-Sapphire laser (Coherent Inc, CA) and water immersion 20x/0.95 N.A. objective (Olympus, Japan). Images were taken using 850nm laser light with power of 15mW at the objective.
3
Culturing Caco-2 and HEK293 Cells
4
Quantifying F-Actin and G-Actin in NOX5β-Expressing Cells
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HEK293 cells (ATCC) were cultured at equal cell densities, transfected with NOX5β or empty vector DNA using methods described above, and lysed with F-Actin/G-Actin In Vivo Assay Biochem Kit (Cytoskeleton) according to the instructions provided (Alternatively, HEK293 cells (ATCC) and HEK293 cells stably expressing NOX5β were used). After lysing, the samples were homogenized using a 25G needle and incubated at 37°C for 10 min 100 μL of lysate were placed into 1.5 mL tubes and centrifuged at a speed of 350 × g at room temperature for 5 min. The supernatant was removed and placed into a labeled ultracentrifuge tube and centrifuged at 100,000 × g at 37°C for 1 h using Beckman-Coulter Optima MAX-TL Ultracentrifuge. Supernatant was gently removed and placed into labeled 1.5 mL tubes. Pellet was dissolved in 100 μL of F-actin depolymerization buffer (provided in kit) for 1 h on ice while pipetting up and down several times every 15 min. Samples were analyzed via western blot analysis as described above and band densities determined using Empiria Studio 2.2.
5
Optimizing shRNA Silencing Plasmids
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Mouse Rps6ka2 or rpS6 gene shRNA sequences were designed according to the cDNA sequence using online software (http://bioinfo.clontech.com/rnaidesigner/frontpage.jsp ). Following synthesis and annealing, four double-stranded oligonucleotides (dsOligo) were cloned into the pDC316-gfp-U6 plasmid (Miaoling Bioscience & Technology, Wuhan, China), and the sequences were confirmed by PCR and DNA sequencing. Real-time PCR and western blotting were used to screen the most effective pDC316-gfp-Rps6ka2-shRNA or pDC316-gfp-rpS6-shRNA plasmid in HEK293 cells (ATCC, USA), and the most effective plasmid was packaged into a recombinant adenovirus AD-Rps6ka2-shRNA or AD-rpS6-shRNA with adenovirus packing materials in HEK293 cells. The adenovirus titer was determined using a hole-by-dilution titer assay. The silencing effect of AD-Rps6ka2-shRNA in CSPCs of MRL/MpJ mice or AD-rpS6-shRNA in CSPCs of CBA mice was validated by western blotting.
6
Stable Transfection and Transient Transfection of Cell Lines
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U937 cells, DC6 and AD3 derived clones, were cultured at 37°C in a humidified atmosphere of 5% CO 2 in RPMI 1640 medium, and HEK293 cells were cultured in Dulbecco´s modified Eagle´s medium (DMEM); all supplemented with 10% FBS and 50 g/ml gentamicin. DC6 and AD3 cell lines were obtained by U937 stable transfection with pcDNA3-HA-dynaminK44A or pcDNA3-β 1 arrestin (319-418) respectively, and were previously characterized (Fernandez et al., 2008) (link). U937 and HEK293 cells were purchased from ATCC.
For transient transfection, HEK293 cells were grown to 80-90% confluency and the cDNA constructs were transfected using K2 Transfection System. The transfection protocol was optimized as recommended by the supplier (Biontex, Munich, Germany).
Usually, assays were performed 48 h after transfection. The human histamine H 1 receptor (H 1 R) and histamine H 2 receptor (H 2 R) were subcloned previously in our laboratory in pCEFL and pCEFLHA, respectively (Notcovich et al., 2010; (link)Shayo et al., 2001) .
For transient transfection, HEK293 cells were grown to 80-90% confluency and the cDNA constructs were transfected using K2 Transfection System. The transfection protocol was optimized as recommended by the supplier (Biontex, Munich, Germany).
Usually, assays were performed 48 h after transfection. The human histamine H 1 receptor (H 1 R) and histamine H 2 receptor (H 2 R) were subcloned previously in our laboratory in pCEFL and pCEFLHA, respectively (Notcovich et al., 2010; (link)Shayo et al., 2001) .
7
Transient Transfection of TRPA1 Variants in HEK293 Cells
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Naïve untransfected HEK293 cells (American Type Culture Collection, Manassas, VA, USA; ATCC® CRL‐1573™) were cultured according to the manufacturer's instructions. HEK293 cells were transiently transfected with the cDNAs (1 μg) codifying for wild‐type (wt‐hTRPA1) or mutant 3C/K‐Q human TRPA1 (C619S, C639S, C663S, K708Q; 3C/K‐Q hTRPA1‐HEK293)23 using the jetPRIME transfection reagent (Poliyplus‐transfection® SA, Strasburg, France) following the manufacturer's protocol. HEK293 cells stably transfected with cDNA for human TRPA1 (hTRPA1‐HEK293), or with cDNA for human TRPV1 (hTRPV1‐HEK293), or with cDNA for human TRPV4 (hTRPV4‐HEK293) were cultured as previously described.28 Human foetal lung fibroblasts (IMR90; American Type Culture Collection; ATCC® CCL‐186™), which express the native TRPA1 channel, were cultured as previously described.29 Cells were plated on glass‐coated (poly‐l ‐lysine, 8.3 μmol/L) coverslips and cultured for 1‐2 days before being used for recordings.
8
Expression and Purification of Complement Proteins
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FHR-3 with a Strep-tag II was expressed in HEK293 cells (American Type Culture Collection) as previously described (10 (link)).
FHR-1 was transiently expressed in HEK293 cells. For this, the CFHR1 gene was cloned into expression vector pEXPR-IBA103 containing a c-terminal Strep-tag II (IBA) by using specific in-house-designed primer pairs (Metabion,Supplementary Table 1
FHR-1 was transiently expressed in HEK293 cells. For this, the CFHR1 gene was cloned into expression vector pEXPR-IBA103 containing a c-terminal Strep-tag II (IBA) by using specific in-house-designed primer pairs (Metabion,
9
Induction of Phosphorylated TDP-43 in HEK293 Cells
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HEK293 cells
(ATCC, Manassas,
VA) were cultured in 6-well dishes under standard culture conditions
in Dulbecco Modified Eagle Medium (DMEM), 10% defined fetal bovine
serum (FBS), and penicillin (50 IU/mL)–streptomycin (50 mg/mL).
CK-1δ inhibitors were diluted in dimethyl sulfoxide (DMSO),
and 8 μL of inhibitor + DMSO or DMSO alone (control) were added
to cells at the indicated concentrations. After 1.5 h of exposure
to the CK-1δ inhibitor alone, ethacrynic acid was added at a
final concentration of 150 μM and cells were incubated for 4
h to induce phosphorylation of endogenous TDP-43. Cells were harvested,
washed in phosphate buffered saline, and snap frozen prior to preparation
for immunoblot. Cell lysates were loaded and resolved on precast 4–15%
gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis
gels and transferred to polyvinylidene difluoride membrane as recommended
by the manufacturer (Bio-Rad, Hercules, CA). On immunoblots, phosphorylated
human TDP-43 (pS409/410) was detected with a monoclonal antibody (TIP-PTD-M01,
Cosmo Bio, Carlsbad, CA). Actin (load control) was detected with a
monoclonal antibody (A4700, Sigma-Aldrich, St. Louis, MO).
(ATCC, Manassas,
VA) were cultured in 6-well dishes under standard culture conditions
in Dulbecco Modified Eagle Medium (DMEM), 10% defined fetal bovine
serum (FBS), and penicillin (50 IU/mL)–streptomycin (50 mg/mL).
CK-1δ inhibitors were diluted in dimethyl sulfoxide (DMSO),
and 8 μL of inhibitor + DMSO or DMSO alone (control) were added
to cells at the indicated concentrations. After 1.5 h of exposure
to the CK-1δ inhibitor alone, ethacrynic acid was added at a
final concentration of 150 μM and cells were incubated for 4
h to induce phosphorylation of endogenous TDP-43. Cells were harvested,
washed in phosphate buffered saline, and snap frozen prior to preparation
for immunoblot. Cell lysates were loaded and resolved on precast 4–15%
gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis
gels and transferred to polyvinylidene difluoride membrane as recommended
by the manufacturer (Bio-Rad, Hercules, CA). On immunoblots, phosphorylated
human TDP-43 (pS409/410) was detected with a monoclonal antibody (TIP-PTD-M01,
Cosmo Bio, Carlsbad, CA). Actin (load control) was detected with a
monoclonal antibody (A4700, Sigma-Aldrich, St. Louis, MO).
10
Isolation and Immortalization of Mouse Embryonic Fibroblasts
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MEFs were prepared from mice of the indicated genotypes at E13.5–E14.5 after coitus using a standard method51 . Briefly, a pregnant mouse was euthanized, and the uterus was removed from the abdomen. After removing the yolk sac and placenta from the embryo, the embryo was decapitated, and the limbs, tail, and internal organs were removed. The remaining body was minced with scissors and then incubated with trypsin/EDTA (0.25%)-PBS at 37 °C for 30 min. After adding 10% FBS-DMEM to cell suspensions to neutralize trypsin, cell suspensions were passed through a nylon mesh. Resultant cell suspensions were plated on a 10 cm dish and expanded until cells became confluent. Then cells were stocked in liquid nitrogen. MEFs below ten passages were used as primary MEFs (pMEFs) for experiments. Wild-type and Mlkl−/– MEFs were immortalized by transfection with a pEF321-T vector that encodes SV40 large T antigen52 (link). HEK293 cells were obtained from ATCC. HEK293 cells and MEFs were maintained in a DMEM medium containing 10% fetal bovine serum (FBS).
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