Nih 3t3 flp in cells

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The NIH/3T3 Flp-In cells are a stable cell line derived from NIH/3T3 mouse embryonic fibroblasts, engineered to contain a Flp recombination target (FRT) site. This cell line allows for the rapid and efficient generation of cell lines expressing a gene of interest under the control of a constitutive promoter through Flp-mediated recombination.

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NIH 3T3 Flp-In (5 citations) Flp-In-3T3 (2 citations)

7 protocols using nih 3t3 flp in cells

Culturing and Transfecting HEK293T and NIH 3T3 Cells

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Human embryonic kidney 293T cells (293 cells; American Type Culture Collection CRL-3216) were cultured in DMEM supplemented with 10% calf serum (CS) at 37°C and 5% CO₂. NIH 3T3 Flp-In cells (Invitrogen) were cultured in DMEM with 10% FCS. All cell lines were sub-cultured every 2–3 days and were regularly checked by PCR according to reference (54 (link)) for the absence of mycoplasm contaminations. For transfection of HEK293T cells, standard calcium phosphate co-precipitation with 1–5 µg of plasmid DNA for each 10 cm culture dish was used. NIH 3T3 Flp-In cells were transfected using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Cells were employed in infection experiments 24–48 h after transfection.

Shi Y., Muenzner P., Schanz-Jurinka S, & Hauck C.R. (2024). The phosphatidylinositol-5′ phosphatase synaptojanin1 limits integrin-mediated invasion of Staphylococcus aureus. Microbiology Spectrum, 12(4), e02006-23.

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Culturing Cell Lines for GPR161 Studies

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T-REx-293 (Invitrogen), IMCD3 Flp-In, and Phoenix A (PhA) cells (National Gene Vector Biorepository) were cultured in DMEM high glucose (Sigma-Aldrich; supplemented with 10% FBS [Sigma-Aldrich], 0.05 mg/ml penicillin, 0.05 mg/ml streptomycin, and 4.5 mM glutamine). NIH 3T3 Flp-In cells (Invitrogen) were grown in DMEM high glucose (supplemented with 10% bovine calf serum [Sigma-Aldrich], 0.05 mg/ml penicillin, 0.05 mg/ml streptomycin, and 4.5 mM glutamine). NIH 3T3 Flp-In cell lines stably expressing wild type/mutant Gpr161^GFP (Gpr161 followed by Speptide-PrecissionS-EGFP [LAP5]), and untagged receptor constructs were generated using transfection or retroviral infection and antibiotic selection (Mukhopadhyay et al., 2013 (link)). ARPE-19 cells were grown in DMEM F12 (Sigma-Aldrich) media with 10% FBS (Sigma-Aldrich), 0.05 mg/ml penicillin, 0.05 mg/ml streptomycin, and 4.5 mM glutamine. Stable CAMYEL T-REx-293 cells (Invitrogen) inducibly coexpressing untagged mouse Gpr161 were generated by transfection of plasmids and antibiotic selection. Transfection of plasmids was done with either Polyfect (QIAGEN) or Bio-T (BioLand Scientific).

Pal K., Hwang S.H., Somatilaka B., Badgandi H., Jackson P.K., DeFea K, & Mukhopadhyay S. (2016). Smoothened determines β-arrestin–mediated removal of the G protein–coupled receptor Gpr161 from the primary cilium. The Journal of Cell Biology, 212(7), 861-875.

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Lentiviral Reporter Assay for Hedgehog Signaling

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A minimal promoter and 8xGLI-binding-site (GBS) sequences were inserted upstream of GFP within a pRRL.sin-18.PPT.GFP.pre lentiviral plasmid vector (Monje et al., 2011 (link)), and prepared lentiviruses were used to infect NIH/3T3-Flp-In cells (Life Technologies) in the presence of 4 µg/ml polybrene (Sigma). Cells were grown to confluence prior to serum starvation in the presence of 200nM SAG (Enzo Life Sciences) for 24 hours. GFP-expressing cells were single cell sorted into a 96-well plate using FACSAria II (BD Biosciences), and multiple single cell-derived clones were analyzed for SAG-induced GFP expression. For all reporter assay experiments, a specific clone with low basal GFP expression and high SAG-induced GFP expression was used. 40000 cells were seeded into one well of Lab-Tek 8-well chamber (Thermo Scientific), and cells were induced for quiescent state by culturing in serum starvation media (DMEM containing 2mM GlutaMAX) for 48 hours. Afterwards, cells were treated with serum starvation media containing 20% FBS or 200nM SAG (Enzo Life Sciences), in the presence or absence of 1µM Vismodegib (LC labs) for 8 hours or 24 hours. Cells were then fixed with 4% paraformaldehyde at room temperature for 10 mins prior to imaging of reporter GFP expression.

Phua S.C., Chiba S., Suzuki M., Su E., Roberson E.C., Pusapati G.V., Setou M., Rohatgi R., Reiter J.F., Ikegami K, & Inoue T. (2017). Dynamic remodeling of membrane composition drives cell cycle through primary cilia excision. Cell, 168(1-2), 264-279.e15.

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Serum-starved HEK-293T and NIH/3T3 Cells

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HEK-293T cells (ATCC), and NIH/3T3 Flp-In cells (Life Technologies), including derivative stable clones were cultured in media composed of DMEM (high glucose), 10% fetal bovine serum (FBS; Thermo Fisher Scientific), 1x GlutaMAX, 1x non-essential amino acids, 1x sodium pyruvate, 1x penicillin/streptomycin (all from Life Technologies). Prior to harvesting, the cells were serum-starved in the same media but containing 0.5% FBS for 24 hours with or without SAG (400 nM; Enzo) depending on the experiment.

Markiewicz Ł., Uśpieński T., Baran B., Niedziółka S.M, & Niewiadomski P. (2021). Xpo7 negatively regulates Hedgehog signaling by exporting Gli2 from the nucleus. Cellular signalling, 80.

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Serum-starved HEK-293T and NIH/3T3 Cells

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Markiewicz Ł., Uśpieński T., Niedziółka S.M., & Niewiadomski P. (2020). Xpo7 negatively regulates Hedgehog signaling by exporting Gli2 from the nucleus.

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Stable Cell Lines for EVC Complexes

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NIH/3T3, C3H10T1/2 and HEK293T cells were obtained from ATCC, and Flp-In NIH/3T3 cells for stable cell lines construction from Life Technologies. Stable cell lines expressing tagged EVC, EVC2, EFCAB7 and IQCE (or their mutants) were produced by site-specific recombination into a single site in the genome using the Flp-In system (Life Technologies).

Pusapati G.V., Hughes C.E., Dorn K.V., Zhang D., Sugianto P., Aravind L, & Rohatgi R. (2014). EFCAB7 and IQCE regulate Hedgehog signaling by tethering the EVC-EVC2 complex to the base of primary cilia. Developmental cell, 28(5), 483-496.

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Culturing Mouse Cell Lines

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Mouse Flp-In NIH 3T3 cells (male - Life Technologies) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing; 10% fetal calf serum (FCS), 1% Penicillin/Streptomycin, 25 mM D-Glucose, 4 mM L-glutamine, 1 mM sodium pyruvate and 100 μg/ml Zeocin (Gibco). Mouse E14Tg2A embryonic stem cells (from mouse strain 129/Ola) were cultured in Glasgow’s modified Eagle’s medium (GMEM BHK-21) containing 10% FBS, 1% Sodium Pyruvate, 1% MEM non-essential amino acids, 2mM Glutamine, 0.1mM 2-Mercaptoethanol and 106 U/L LIF (prepared in house). Mouse embryonic fibroblasts were isolated from E12.5 embryos and maintained in Opti-mem (GIBCO), 10% v/v FCS, 1% v/v Penicillin/Streptomycin, 0.1mM 2-Mercaptoethanol (Sigma). All cell lines were maintained in a humidified incubator at 37°C supplied with 5% CO₂ in air.

Ford M.J., Yeyati P.L., Mali G.R., Keighren M.A., Waddell S.H., Mjoseng H.K., Douglas A.T., Hall E.A., Sakaue-Sawano A., Miyawaki A., Meehan R.R., Boulter L., Jackson I.J., Mill P, & Mort R.L. (2018). A Cell/Cilia Cycle Biosensor for Single-Cell Kinetics Reveals Persistence of Cilia after G1/S Transition Is a General Property in Cells and Mice. Developmental Cell, 47(4), 509-523.e5.

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