Human renal cell carcinoma cell lines 786-O (Cat# CRL-1932) and 769-P (Cat# CRL-1933) were obtained from American Type Culture Collection (ATCC). HEK293FT was purchased from ThermoFisher Scientific (R70007). Short Tandem Repeat (STR) analyses were performed to authenticate the identity of each Cell line used in this article. The 786-O and 769-P cells were cultured in RPMI-1640 medium (#SH30809.01; HyClone). The HEK293FT cells were cultured in Dulbecco’s Modified Eagle’s medium (#C11995599CP; Gibco). All three cell lines were supplemented with 10% fetal bovine serum (#10099-141C; Gibco), 1% penicillin-streptomycin (#SV30010; Hyclone), and 1% GlutaMaxTM Supplement (#35050-061; Gibco) and grown in a humidified 5% CO2 atmosphere at 37 °C.
Hek293ft
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The HEK293FT is a human embryonic kidney cell line that is designed for high-efficiency transient transfection experiments. It is derived from the HEK293 cell line and has been engineered to express the SV40 large T antigen, which enhances transfection efficiency and protein expression.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (32 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (26 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (22 mentions)
L glutamine, by Thermo Fisher Scientific (15 mentions)
Mcf 7, by American Type Culture Collection (13 mentions)
Streptomycin, by Thermo Fisher Scientific (9 mentions)
Penicillin, by Thermo Fisher Scientific (9 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (8 mentions)
Glutamax, by Thermo Fisher Scientific (8 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (8 mentions)
Hek293t, by American Type Culture Collection (7 mentions)
Hek293ft cells, by Thermo Fisher Scientific (7 mentions)
Hek293ft, by American Type Culture Collection (7 mentions)
Hek293t, by Thermo Fisher Scientific (6 mentions)
Fetal bovine serum (fbs), by Merck Group (6 mentions)
Hek293, by American Type Culture Collection (6 mentions)
Hepg2, by American Type Culture Collection (6 mentions)
Rpmi 1640, by Thermo Fisher Scientific (6 mentions)
Mda mb 231, by American Type Culture Collection (6 mentions)
Vero cell, by American Type Culture Collection (6 mentions)
185 protocols using hek293ft
1
Culturing Renal Cell Carcinoma Lines
2
Cell Culture Maintenance Protocols
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Human DU145, PC3 were from DSMZ and HEK293FT from Thermofisher. DU145, PC3 and HEK293FT were cultured in DMEM with L-Glutamine and pyruvate (Gibco) supplemented with 10% FBS (Gibco) and 1% Penicillin/streptomycin (Gibco) and maintained in a humidified atmosphere at 37°C and 5% CO2. Cell cultures were tested for mycoplasma monthly and maintained mycoplasma-free.
3
Stem Cell Culture and Lentiviral Transduction
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Human embryonic stem cell line H9 was obtained from WiCell Research Institute, Inc. and cultured with mTeSR1 medium (STEMCELL, USA). Human fibroblasts were used between passages 3 to 12. The HEK 293 FT and HEK 293T cell lines were purchased from Thermo Scientific. Human fibroblasts and HEK 293 FT were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS) (Hyclone) and 1% penicillin-streptomycin (Pen/Strep) (Gibco) at 37 °C with 5% CO2. The lentivirus production and transduction were performed as we previously described (Kim et al., 2019 (link)). All cell lines were routinely checked for mycoplasma by a PCR detection kit.
4
Cell Line Preparation and Culture
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The human GBC cell line GBC-SD was purchased from the Shanghai Institutes for Biological Sciences (Shanghai, China). The CC cell line QBC-939 was obtained from Prof. Shuguang Wang (The Third Military Medical University, China). Human embryonic kidney 293 cells (HEK293FT) were purchased from Invitrogen (Gaithersburg, MD, USA). Liquid nitrogen stocks were made upon receipt and maintained until the start of each study. Cells were used for no >3 months after being thawed. GBC-SD and HEK293FT were cultured in DMEM medium, and QBC-939 cells was cultured in RPMI-1640 medium, with all media containing 10% FBS, 100 units/ml penicillin and 100 mg/ml streptomycin (Gibco, Grand Island, NY). Cells were incubated at 37 °C in a humidified incubator under 5% CO2 and tested negative for mycoplasma infection by LookOut® mycoplasma qPCR detection kit (Sigma, St. Louis, MO, USA).
5
Generation of Cas13d-expressing Cell Lines
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HEK293FT cells were acquired from Thermo Fisher Scientific (R70007) and A375 cells were acquired from ATCC (CRL-1619). HEK293FT and A375 cells were maintained at 37°C with 5% CO2 in D10 media: DMEM with high glucose and stabilized L-glutamine (Caisson DML23) supplemented with 10% fetal bovine serum (Serum Plus II Sigma-Aldrich 14009C) and no antibiotics.
To generate doxycycline-inducible RfxCas13d-NLS HEK293FT and A375 cells, we transduced cells with a RfxCas13d-expressing lentivirus at low MOI (<0.1) and selected with 5μg/mL Blasticidin S (ThermoFisher A1113903). Single cell colonies were picked after by sparse plating. Clones were screened for Cas13d expression by western blot using mouse anti-FLAG M2 antibody (Sigma F1804).
For the GFP tiling screen RfxCas13d-expressing cells were transduced with pLentiEGFPdestabilized lentivirus at low MOI (<0.1) and selected with 100μg/ml Hygromycin B (ThermoFisher 10687010) for 2 days. Single-cell colonies were grown by sparse plating. Resistant and GFP-positive clonal cells were expanded and screened for homogenous GFP expression by flow cytometry.
To generate doxycycline-inducible RfxCas13d-NLS HEK293FT and A375 cells, we transduced cells with a RfxCas13d-expressing lentivirus at low MOI (<0.1) and selected with 5μg/mL Blasticidin S (ThermoFisher A1113903). Single cell colonies were picked after by sparse plating. Clones were screened for Cas13d expression by western blot using mouse anti-FLAG M2 antibody (Sigma F1804).
For the GFP tiling screen RfxCas13d-expressing cells were transduced with pLentiEGFPdestabilized lentivirus at low MOI (<0.1) and selected with 100μg/ml Hygromycin B (ThermoFisher 10687010) for 2 days. Single-cell colonies were grown by sparse plating. Resistant and GFP-positive clonal cells were expanded and screened for homogenous GFP expression by flow cytometry.
6
Inducing Arc Expression in Neuroblastoma Cells
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Human embryonic kidney 293FT (HEK293FT) and SH-SY5Y neuroblastoma cells were obtained from Thermo Fisher Scientific (Waltham, MA) and ATCC (Manassas, VA), respectively. Cells were maintained in Dulbecco’s modified Eagle medium (DMEM/high-glucose, Sigma-Aldrich, St. Louis, MO) supplied with 10% FBS (Sigma-Aldrich) and 100 U/ml Penicilin-Streptomycin (Thermo Fisher Scientific) at 37 ºC in a humidified incubator with 5% CO2. HEK293FT cells were transfected using Lipofectamine™ 2000 Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. For induction of endogenous Arc expression, SH-SY5Y cells were stimulated with 100 μM carbachol (CCh) for 1 h at 37 ºC in a humidified incubator with 5% CO2 [36 (link)].
7
HEK Cell Transfection with Lipofectamine
8
Cell Line Authentication and Mycoplasma Screening
9
Cell Culture and Differentiation Protocols
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The human monocytic leukaemia cell line THP-1 (ATCC: TIB202TM) was cultured in RPMI (Gibco) medium supplemented with 10% fetal bovine serum (Sigma), 1 mM pyruvate, 200 mM l -glutamine, 100 U ml−1 penicillin and 100 mg ml−1 streptomycin in an incubator at 37°C with 5% CO2. These cells were differentiated to macrophages with 40 ng ml−1 PMA (Sigma) for 72 h. Afterwards, the cells were washed with PBS and incubated with fresh medium for more than 72 h. Mouse macrophage leukaemia cell line RAW 264.7 (ATCC: TIB-71) and human embryonic kidney cell line HEK-293FT (Life Technologies) were cultured in DMEM (Gibco) medium supplemented with 10% fetal bovine serum, 100 U ml−1 penicillin and 100 mg ml−1 streptomycin in an incubator at 37°C with 5% CO2. HEK-293FT cells were maintained in medium containing 500 µg ml−1 Geneticin.
10
Cell Culture Conditions and Transfection
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HEK293FT (Life Technologies, Carlsbad, CA) and HeLa M cells (provided by P. De Camilli, Yale University, New Haven, CT) were grown in high-glucose DMEM (+l -glutamine), 10% fetal bovine serum, and 1% penicillin/streptomycin supplement (Invitrogen, Carlsbad, CA). Where indicated, cells were starved for 90 min by incubation with amino acid–free RPMI media (US Biological, Swampscott, MA). Amino acid refeeding was performed with 1× MEM amino acid supplement (Invitrogen) added to starvation medium for 15 min. Transfections were performed with 500 ng of DNA, 100 μl of OptiMEM (Invitrogen), and 1.5 μl of FuGENE 6 transfection reagent (Promega, Madison, WI) per 35-mm dish and scaled up proportionately for larger and smaller dishes. siRNA transfections were performed with 5 μl of RNAiMAX (Invitrogen), 500 μl of OptiMEM (Invitrogen), and 7.5 μl of 20 μM siRNA stock suspended in 100 mM KAc and 30 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.5. We used 250,000 HEK 293FT cells per 35-mm dish for siRNA transfection and incubated them for 2 d posttransfection before experiments. siRNA sequences purchased from Integrated DNA Technologies (IDT, Coralville, IA) are summarized in Supplemental Table S1.
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