HEK293A (Thermo Fisher Scientific) and Surf4KO HEK293A cells (see following subsection, Generation of Surf4KO HEK293A cells) were maintained in Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Sigma), GlutaMAX (2 mM, Thermo Fisher Scientific), penicillin 100 (IU/mL), and streptomycin (100μg/mL). Expression constructs were transfected into cells using Lipofectamine 2000 reagent (Thermo Fisher Scientific). Briefly, 5×105 cells/well were plated into 6-well plates. After overnight culture (60%–80% confluent), cells were transfected with 2μg of plasmid DNA preincubated with 4μL Lipofectamine 2000 in Opti-MEM (Thermo Fisher Scientific). Transient expression of proteins was analyzed 16–24 hr posttransfection as noted.
Hek293a
Manufactured by Thermo Fisher Scientific
Sourced in United States
HEK293A is a well-established cell line derived from human embryonic kidney cells. It is commonly used as a host cell line for the expression of recombinant proteins and the production of viruses in various research and application areas.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (12 mentions)
Hek293t, by American Type Culture Collection (8 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (7 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (7 mentions)
Streptomycin, by Thermo Fisher Scientific (6 mentions)
Fetal bovine serum (fbs), by Merck Group (5 mentions)
Hek293a cells, by Thermo Fisher Scientific (5 mentions)
Penicillin, by Thermo Fisher Scientific (5 mentions)
Glutamax, by Thermo Fisher Scientific (4 mentions)
Hek293t, by Thermo Fisher Scientific (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Glutamine, by Thermo Fisher Scientific (3 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (3 mentions)
Pentr d topo, by Thermo Fisher Scientific (3 mentions)
Pad cmv v5 dest adenoviral vector, by Thermo Fisher Scientific (3 mentions)
Hacat, by American Type Culture Collection (2 mentions)
Hepg2, by American Type Culture Collection (2 mentions)
Fetal bovine serum (fbs), by Lonza (2 mentions)
Penicillin streptomycin, by Corning (2 mentions)
68 protocols using hek293a
1
HEK293A Cell Transfection Protocol
2
Cell Culture and Transfection Protocols
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A431 cells (ATCC CRL-1555), U2OS cells (kindly provided by Marikki Laiho at John Hopkins University, USA), and HEK293A cells (Invitrogen R70507) were cultured in DMEM (high-glucose, Lonza/Gibco), and A549 cells (ATCC CCL-185) in F-12 Nutrient Mixture (Gibco), supplemented with 10% FBS, penicillin/streptomycin (100 U ml−1 each), L‐glutamine (2 mM) at 37 °C in 5% CO2. Cells were tested negative for Mycoplasma using PCR detection. Cells were seeded for 16 h in 12-well and transfected at 80–95% confluence using Lipofectamine LTX with PLUS Reagent (Invitrogen 15338–100), typically with 1.0 µg plasmid(s) per 1.0 µl of PLUS Reagent (1.5 µl for HEK293A), 2.0 µl of Lipofectamine LTX (3.0 µl for HEK293A), and 4.0 × 105 (A431 and HEK293A) or 3.0 × 105 (U2OS and A549) cells per well.
3
Cell Culture and Virus Propagation
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Vero (CCL-81) cells were obtained from the American Type Culture Collection (ATCC, USA). Rhabdomyosarcoma (RD) cells and Madin-Darby Canine Kidney (MDCK) cells were obtained from the Bioresource Collection and Research Center (BCRC), Hsinchu, Taiwan. Human embryonic kidney 293 adherent cells (HEK293A) were purchased from Invitrogen (Cat. R70507). Vero, RD and MDCK cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) with 10% fetal bovine serum (FBS). HEK293A were recovered in DMEM+10%FBS and adapted into serum-free medium using FreeStyle 293 medium (Gibco) with 1x cell culture media supplement (ITSE + A, InVitria) in roller bottle culture and BalanCD HEK293 medium (FUJIFILM Irvine Scientific) in suspension culture. The cell density was counted by a hemocytometer. The CVA10 (strain M2014) and CVA6 (strain M0746) were obtained from National Cheng Kung University Hospital, Taiwan. The EV-A71 (strain E59) was obtained from the Center of Disease Control, Taiwan. These virus stocks were prepared from the supernatants of infected RD cells at 3 days post-infection (DPI). The titers of virus were determined by the median tissue culture infectious dose (TCID50) assay.
4
Isolation and Cultivation of PBMCs and Cell Lines
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Peripheral Blood Mononuclear Cells (PBMCs) were a gift from Dr. June Kant-Mitchell; these cells had been collected from healthy volunteers, who had provided written informed consent according to a previously approved protocol at the University of Texas at El Paso (UTEP), and kept frozen as 1 mL aliquots at approximately 1 × 106 cells per mL at − 80 °C, with each vial corresponding to cells from one volunteer only. Approval for the use of the PBMCs was obtained from the Institutional Review Board (IRB) Committee at UTEP as well as from the granting institution, U.S. Army Medical Research and Development Command, Office of Research Protections, Human Research Protection Office. When needed, the PBMCs were thawed and directly used for RNA purification as described below. A total of three different vials, from three different individuals, were used in these studies. Human embryonic kidney cells (HEK293A) were from Invitrogen (ThermoFisher Scientific, Inc., Waltham, MA). A549 and Calu-3 cells were from ATCC (American Type Culture Collection). HEK293A, A549, and Calu-3 cells were grown at 37 °C, 5% CO2, in 1 × Complete Medium consisting of 1 × DMEM containing high glucose, pyruvate, and GlutaMAX™ (Gibco™, ThermoFisher Scientific, Inc.), supplemented with 10% Fetal Bovine Serum.
5
Cell culture protocol for multiple lines
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Eighteen different cell lines were used in this study (Table 1 ), which included, HBMEC (ScienCell); SK-N-SH (ATCC HTB-11); HaCaT (ATCC PCS-200-011); Rhadomyosarcoma (ATCC CCL-136); SJCRH30 (ATCC CRL-2061); A549 (ATCC CCL-185); HUH 7 (Dr Priscilla Yang, Harvard Medical School, USA); HUH 7.5 (Dr Yoichi Suzuki, Osaka Medical College Hospital, Japan); HepG2 (ATCC HB-8065); HEK293A (Life Technologies); HEK293T (Life Technologies); HeLa CCL2 (ATCC CRM-CCL-2); Vero (ATCC CCL-81); Vero C1008 (ATCC CRL-1586); Baby Hamster Kidney (BHK) 21 (ATCC CCL-10); Chinese Hamster Ovary (CHO) (ATCC CCL-61); C2C12 (ATCC CRL-1772) and Aedes. albopictus C6/36 (ATCC CRL-1660). The growth medium used are from Sigma-Aldrich Corp., St Louis, MO, USA. Cell lines 1 to 17 (Table 1 ) were cultured in RPMI and DMEM 10% fetal bovine serum (FBS) (Capricorn Scientific, South America), at 37°C in the presence of 5% CO2. In addition, C6/36 cells were cultured in L-15 media supplemented with 10% heat-inactivated FBS (Capricorn Scientific, South America) at 28°C. HSMM cells were cultured in skeletal muscle cell growth medium (SkGM) (Lonza) supplemented with growth factors and 10% FBS (Lonza) at 37°C with 5% CO2.
6
Culturing HEK-293A Cells in DMEM
7
HTS with Various Cell Lines
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Four cell lines were initially assessed for the HTS: rat insulinoma cells (Ins1 823/13), mouse insulinoma cells (βTC3), human hepatoma cells (HepG2) and human colon cancer cells (HCT116). The human hepatoma cell line used for these studies, HepG2 (ATCC), was cultured in EMEM medium supplemented with 10% FCS, 1% penicillin-streptomycin. The adenovirus packaging cell line, HEK-293A (Life Technologies) was cultured in DMEM medium supplemented with 10% FCS, and 1% penicillin-streptomycin, and 1x MEM containing non-essential amino acids. Cell lines were not validated by genomic testing, and we not tested for mycoplasma.
8
HEK293A Cell Culture Protocol
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Human embryonic kidney cells (HEK293A) were purchased from Life Technologies and maintained in DMEM (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Corning). Cells were passaged at 75% confluency.
9
HTS with Various Cell Lines
10
Culturing HEK 293A Cells in DMEM
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HEK 293A (Life technologies, #R70507) cells were cultured in DMEM (Gibco, #21969-035) supplemented with 10% FCS (Merck, #S0115), 1% Penicillin-Streptomycin (10,000 U/mL, Gibco, #15140-122) and 1% glutamine (Gibco, #25030-024) at 37 °C, 5% CO2.
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