293ft cell

Manufactured by Thermo Fisher Scientific

Sourced in United States, Japan, Germany, China

293FT cells are a human embryonic kidney cell line that are commonly used in a variety of cell biology research applications. These cells are derived from the 293 cell line and have been engineered to express the large T antigen of the SV40 virus, which enhances their ability to support the replication of viral vectors. 293FT cells are widely used for the production and amplification of various types of viral vectors, including lentivirus, adenovirus, and adeno-associated virus.

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405 protocols using 293ft cell

Lentiviral Particle Production in 293FT Cells

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293FT cells (ThermoFisher Scientific, Cat# R70007) were used to
generate lentiviral particles through co-transfection of the packaging
vectors pCMV-dR8.2 dvpr (Addgene, Plasmid # 8455) and pCl-VSVG (Addgene,
Plasmid # 1733) with the appropriate overexpression, shRNA, or other
plasmid. 293FT cells were seeded at a density of 1.2 million cells in DMEM,
high glucose (ThermoFisher Scientific, Cat# 11995073) in 10% Fetal Bovine
Serum (ThermoFisher Scientific, Cat# 26140079 with 1%
Penicillin-Streptomycin (ThermoFisher Scientific, Cat # 15140122). Cells
were incubated for 24 hours prior to transfection. Transfection was
performed using LipoD293^™ In Vitro DNA Transfection
Reagent (SignaGen Laboratories, Cat # SL100668) according to the
manufacturer’s instructions. Briefly, 5μg each of the
packaging plasmids and the plasmid of interest were combined into a tube,
the transfection reagent was diluted and added followed by a 15 minute
incubation. The transfection mixture was then added to the 293FT cells.
Media was changed after 16 hours. Virus was collected 48 hours after media
change and concentrated using the Lenti-X Concentrator (Clontech Takara Bio
USA, Cat # 631232) according to the manufacturers instructions. Viral
supernatants were centrifuged at 1,500 x g for 45 minutes and viral pellets
were frozen at −80°C for future use.

Xie Q., Wu T.P., Gimple R.C., Li Z., Prager B.C., Wu Q., Yu Y., Wang P., Wang Y., Gorkin D.U., Zhang C., Dowiak A.V., Lin K., Zeng C., Sui Y., Kim L.J., Miller T.E., Jiang L., Lee-Poturalski C., Huang Z., Fang X., Zhai K., Mack S.C., Sander M., Bao S., Kerstetter-Fogle A.E., Sloan A.E., Xiao A.Z, & Rich J.N. (2018). N6-methyladenine DNA Modification in Glioblastoma. Cell, 175(5), 1228-1243.e20.

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Authenticated Cell Line Maintenance and Characterization

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MCF10A and T47D cells were purchased from ATCC in 2018. Cell lines were authenticated by ATCC prior to purchase using the tandem short repeat (STR) profiling method. MCF10A cells grown in the laboratory were again authenticated by STR profiling using the ATCC Cell Line Authentication Service in 2022. 293FT cells were purchased from Invitrogen in 2018.
293FT cells were maintained in DMEM supplemented with 10% FBS and 1x antibiotic-antimycotic. T47D cells were maintained in RPMI supplemented with 10% FBS and 1x antibiotic-antimycotic. MCF10A cells were maintained in complete MCF10A medium (DMEM/F12 supplemented with 5% horse serum, 20 ng/ml EGF, 10 mg/ml insulin, 0.5 mg/ml hydrocortisone, 0.1 mg/ml cholera toxin and 1X antibiotic/antimycotic). For all experiments, MCF10A cells were grown in EGF-free DMEM/F12 supplemented with 1% charcoal/dextran-stripped serum (CSS), 0.5mg/ml hydrocortisone, 0.1 mg/ml cholera toxin and 1X Antibiotic-Antimycotic. For western blots and 3D Matrigel experiments, T47D cells were grown in phenol red-free IMEM supplemented with 1% CSS. Cell lines were tested for mycoplasma contamination using the Lonza MycoAlert Mycoplasma Detection Kit (Cat# LT07418) in July 2021. All experiments were completed less than two months or three passages after establishing stable cell lines or early passage thawing of the cells.

Marín A., Al Mamun A., Patel H., Akamatsu H., Ye D., Sudhan D.R., Eli L., Marcelain K., Brown B.P., Meiler J., Arteaga C.L, & Hanker A.B. (2023). Acquired secondary HER2 mutations enhance HER2/MAPK signaling and promote resistance to HER2 kinase inhibition in breast cancer. Cancer research, 83(18), 3145-3158.

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Lentiviral Packaging System for Gene Transduction

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The packaging system for the production of lentiviral consisted of the following vectors: pCMV-ΔR8.91 plasmid, pMD.G (envelope element) and pLKO_AS2/GFP (transfer vector). These vectors were obtained from the National RNAi Core Facility (Taipei, Taiwan) as described previously [15 (link)]. Briefly, 6×10⁵ 293FT cells (Invitrogen) were seeded in a 6-cm culture dish and cultured for 24 hours. A mixture of the 3 vectors (2.5 μg of pCMV-ΔR8.91, 0.3 μg of pMD.G, and 2.5 μg of pLKO_AS2/GFP) was prepared and transfected into 293FT cells using Lipofectamine 2000 (Invitrogen). The virus-containing media were collected at 48 hours after transfection. For lentivirus transduction, human ASCs were seeded in 6-well plates (1×10⁵ per well) in growth media. After overnight incubation, cells were treated with virus-containing media containing 8 μg/mL polybrene (Sigma-Aldrich). The culture medium was removed at 24 hours post-infection and GFP positive cells were enriched (>99%) by FACSAria (BD Biosciences, CA, USA).

Chan E.C., Kuo S.M., Kong A.M., Morrison W.A., Dusting G.J., Mitchell G.M., Lim S.Y, & Liu G.S. (2016). Three Dimensional Collagen Scaffold Promotes Intrinsic Vascularisation for Tissue Engineering Applications. PLoS ONE, 11(2), e0149799.

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Efficient Cre-mediated Gene Deletion in MEFs

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293FT cells (Invitrogen) and MEFs were cultured in DMEM medium (Invitrogen) containing 10% FBS and 1% penicillin-streptomycin (Invitrogen) at 37°C in 5% humidified CO₂ incubators. Lentivirus vector expressing Cre recombinase (Kumar et al., 2008 (link)) (#17408, Addgene) was used for deletion of Ctdp1 in Ctdp1^flox/flox MEFs. The transfection of 293FT cells to produce lentiviral particles expressing Cre (Lenti-Cre) was performed using the calcium phosphatase method and ViraPower system (Invitrogen). For the expression of Cre recombinase, MEFs were transduced with lentivirus in the presence of 10 µg/ml Polybrene (Sigma-Aldrich) for 2 days and then either allowed to recover in fresh medium for the indicated time or followed by selection in 1 µg/ml puromycin to remove uninfected cells. Cells were then used further for various tests.

Qiao F., Law H.C., Krieger K.L., Clement E.J., Xiao Y., Buckley S.M, & Woods N.T. (2021). Ctdp1 deficiency leads to early embryonic lethality in mice and defects in cell cycle progression in MEFs. Biology Open, 10(1), bio057232.

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Cell Line Authentication and Maintenance Protocol

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MCF10A and T47D cells were purchased from ATCC in 2018. Cell lines were authenticated by ATCC prior to purchase using the tandem short repeat (STR) profiling method. MCF10A cells grown in the laboratory were again authenticated by STR profiling using the ATCC Cell Line Authentication Service in 2022. 293FT cells were purchased from Invitrogen in 2018.
293FT cells were maintained in DMEM supplemented with 10% FBS and 1x antibiotic-antimycotic. T47D cells were maintained in RPMI supplemented with 10% FBS and 1x antibiotic-antimycotic. MCF10A cells were maintained in complete MCF10A medium (DMEM/F12 supplemented with 5% horse serum, 20 ng/ml EGF, 10 mg/ml insulin, 0.5 mg/ml hydrocortisone, 0.1 mg/ml cholera toxin and 1X antibiotic/antimycotic). For all experiments, MCF10A cells were grown in EGF-free DMEM/F12 supplemented with 1% charcoal/dextranstripped serum (CSS), 0.5mg/ml hydrocortisone, 0.1 mg/ml cholera toxin and 1X Antibiotic-Antimycotic. For western blots and 3D Matrigel experiments, T47D cells were grown in phenol red-free IMEM supplemented with 1% CSS. Cell lines were tested for mycoplasma contamination using the Lonza MycoAlert Mycoplasma Detection Kit (Cat# LT07418) in July 2021. All experiments were completed less than two months or three passages after establishing stable cell lines or early passage thawing of the cells.

Marín A., Mamun A.A., Patel H., Akamatsu H., Ye D., Sudhan D.R., Eli L., Marcelain K., Brown B.P., Meiler J., Arteaga C.L, & Hanker A.B. (2023). Acquired Secondary HER2 Mutations Enhance HER2/MAPK Signaling and Promote Resistance to HER2 Kinase Inhibition in Breast Cancer. Cancer research, 83(18).

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Androgen-Responsive Prostate Cancer Cell Lines

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Human PCa cell lines LNCaP and PC3 from the ATCC (Manassas, VA) were maintained in RPMI-1640 medium with 10% fetal bovine serum (FBS) or switched to phenol red-free RPMI-1640 with 10% charcoal-stripped FBS (CS-FBS) for androgen-depleted conditions as previously described [99 ]. The 293FT cells from Life Technologies (Grand Island, NY) were maintained in DMEM with 10% FBS. R1881 (methyltrienolone), was obtained from PerkinElmer Life Sciences (Boston, MA). Reporter cell lines Prb-DsRed-LNCaP and Prb-Luc-LNCaP were prepared as described in Supplementary Methods.

Levina E., Ji H., Chen M., Baig M., Oliver D., Ohouo P., Lim C.U., Schools G., Carmack S., Ding Y., Broude E.V., Roninson I.B., Buttyan R, & Shtutman M. (2015). Identification of novel genes that regulate androgen receptor signaling and growth of androgen-deprived prostate cancer cells. Oncotarget, 6(15), 13088-13104.

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LCMV Epitope GP33-41 Lentiviral Vector

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The immunodominant LCMV epitope GP33-41 was synthesized (IDT, Coralville, Iowa) and cloned in pENTR/D-TOPO (Life Technologies). Inserted sequences were confirmed by DNA sequencing (Eton Bioscience, Boston MA). The fragments inserted between attL1 and attL2 sites were further transferred into the plenti-PGK-puro-DEST vector (addgene, Cambridge MA) using Gateway LR Clonase II kit (Life Technologies). Packaging of lentiviral vectors was conducted in 293FT cells (Life Technologies) by transfecting these constructs with pMD2.G and psPAX2 packaging vectors as described previously^{57 (link)}.

Nakashima H., Alayo Q.A., Penaloza-MacMaster P., Freeman G.J., Kuchroo V.K., Reardon D.A., Fernandez S., Caligiuri M, & Chiocca E.A. (2018). Modeling tumor immunity of mouse glioblastoma by exhausted CD8+ T cells. Scientific Reports, 8, 208.

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Production and Purification of AAV2/8 Vectors

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For in-house viral production, 293FT cells (Life Technologies) were maintained as described above in 150mm plates. For each transfection, 8 ug of pAAV8 serotype packaging plasmid, 10 ug of pDF6 helper plasmid, and 6 ug of AAV2 plasmid carrying the construct of interest were added to 1mL of serum-free DMEM. 125 μL of PEI “Max” solution (1mg/mL, pH = 7.1) was then added to the mixture and incubated at room temperature for 5 to 10 seconds. After incubation, the mixture was added to 20 mL of warm maintenance media and applied to each dish to replace the old growth media. Cells were harvested between 48h and 72h post transfection by scraping and pelleting by centrifugation. The AAV2/8 (AAV2 ITR vectors pseudo-typed with AAV8 capsid) viral particles were then purified from the pellet according to a previously published protocol⁵⁰.
High titer and purity viruses were also produced by vector core facilities at Children’s Hospital Boston and Massachusetts Eye and Ear Infirmary (MEEI). These AAV vectors were then titered by real-time qPCR using a customized TaqMan probe against the transgene, and all viral preparations were titer-matched across different batches and production facilities prior to experiments. The purity of AAV vector was further verified by SDS-PAGE.

Ran F.A., Cong L., Yan W.X., Scott D.A., Gootenberg J.S., Kriz A.J., Zetsche B., Shalem O., Wu X., Makarova K.S., Koonin E., Sharp P.A, & Zhang F. (2015). In vivo genome editing using Staphylococcus aureus Cas9. Nature, 520(7546), 186-191.

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Cloning and Expressing Mouse IGFBP-2 in 293FT Cells

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Mouse IGFBP-2 cDNA was amplified from mouse pCMV-SPORT6 (ATCC, Manassas, VA) using a 5′ primer sequence corresponding to nucleotides 89 to 110 of mouse IGFBP-2 (5′-ATGCTGCCGAGATTGGGCGGCC-3′) and a 3′ primer sequence complementary to nucleotides 981 to 1003 (5′-GGGCCCATGCCCAAAGTGTGCAG-3′). After DNA sequencing to confirm that the correct sequence had been amplified, the PCR product was subcloned into pENTR/D-TOPO vector and subsequently transferred into the pLenti6-V5 DEST expression vector using the LR Clonase reaction and following the manufacturer's instructions (Life Technologies, Grand Island, NY). Constructions of RPTPβ and LacZ have been described previously.^{(9 (link))} The constructs contained the correct sequences was verified by DNA sequencing. 293FT cells (Life Technologies, Grand Island, NY) were prepared for generation of virus stocks and CL4 expressing IGFBP-2, RPTPβ and LacZ were established using procedures that have been described previously.^{(12 (link))}

Xi G., Wai C., DeMambro V., Rosen C.J, & Clemmons D.R. (2014). IGFBP-2 directly stimulates osteoblast differentiation. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 29(11), 2427-2438.

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Lentiviral Vector Construction and Titration

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The previously described pSMAL vector modified from the MA1 lentiviral vector to have a Gateway cassette and SFFV promoter was used as the backbone for all generated lentiviral vectors^{49 (link),50 (link)}. HIST1H3H WT/K27M and HIST1H3F WT/K27I with a C-terminal HA-tag and attB sites for Gateway cloning were generated using GeneArt™ DNA Strings Fragments (Life Technologies). Luciferase modified for human expression (Luc2) was amplified out of the pGL4.51[luc2/CMV/NEO] vector (Promega). Genes were cloned into the pDONR221 vector (Invitrogen) using BP Clonase (Invitrogen) and subsequently cloned into the pSMAL vector using LR Clonase (Invitrogen) as per the manufacturer’s instructions. Lentiviral particles were produced in 293FT cells (Life Technologies) as previously. Viral titer was determined by adding serial dilutions of the concentrated viral particles onto 8227 AML cells and the percentage of GFP⁺cells was measured 96 h later by flow cytometry.

Boileau M., Shirinian M., Gayden T., Harutyunyan A.S., Chen C.C., Mikael L.G., Duncan H.M., Neumann A.L., Arreba-Tutusaus P., De Jay N., Zeinieh M., Rossokhata K., Zhang Y., Nikbakht H., Mouawad C., Massoud R., Frey F., Nasr R., El Cheikh J., El Sabban M., Kleinman C.L., Mahfouz R., Minden M.D., Jabado N., Bazarbachi A, & Eppert K. (2019). Mutant H3 histones drive human pre-leukemic hematopoietic stem cell expansion and promote leukemic aggressiveness. Nature Communications, 10, 2891.

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