Nih3t3 cells

Manufactured by American Type Culture Collection

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NIH3T3 cells are a commonly used mouse embryonic fibroblast cell line. They are a well-established model for studying cellular processes such as proliferation, differentiation, and transformation.

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NIH3T3 (543 citations) NIH3T3 mouse fibroblast cell line (5 citations) NIH3T3 mouse embryonic fibroblast cells (3 citations) Mouse NIH3T3 cells (2 citations) NIH3T3 fibroblast cell lines (ATCC® CRL-1658) (2 citations)

243 protocols using nih3t3 cells

Stable transfection of miRNA in NIH/3T3 cells

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NIH/3T3 cells were purchased from ATCC and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Biosera) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (P-S; HyClone) in the presence of 5% CO₂ at 37 °C.
Cells were transfected with 500 ng of either pNeo-miR or pNeo-miR-mP53/1 or pNeo-miR-mP53/2 or pNeo-miR-mP53 in combination with 50 ng of the transposase helper plasmid, using FuGENE® HD transfection reagent (Promega) according to manufacturer’s instructions. Selection of stable transfected cells was performed using neomycin (G418; Biosera).

Kopasz A.G., Pusztai D.Z., Karkas R., Hudoba L., Abdullah K.S., Imre G., Pankotai-Bodó G., Migh E., Nagy A., Kriston A., Germán P., Drubi A.B., Molnár A., Fekete I., Dani V.É., Ocsovszki I., Puskás L.G., Horváth P., Sükösd F, & Mátés L. (2022). A versatile transposon-based technology to generate loss- and gain-of-function phenotypes in the mouse liver. BMC Biology, 20, 74.

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Neonatal Rat Cardiomyocyte Isolation and Manipulation

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Neonatal rat ventricular myocytes (NRVMs) were isolated from 2-day-old rat pups and maintained under standard conditions as previously described [19 (link), 21 (link), 23 (link), 24 (link)]. Isolated NRVMs were cultured for 48 h under quiescent conditions by the inclusion of serum-free DMEM supplemented with 0.1% BSA, 1X ITS, and 1% Pen/Strep prior to experiments. Following quiescence, NRVMs were infected with adenoviral vectors (sh-control or sh-Klf15) for 24 hrs. Following transduction, NRVMs were treated with the exogenous addition of either DMSO (vehicle) or 10 μM WY-14643 for additional 24 hrs. Cells were then harvested, RNA was isolated, and QPCR was performed as described below. NIH-3T3 cells were purchased from ATCC (Manassas, VA) and grown in DMEM supplemented with 10% FBS and 1% Pen/Strep.

Prosdocimo D.A., John J.E., Zhang L., Efraim E.S., Zhang R., Liao X, & Jain M.K. (2015). KLF15 and PPARα Cooperate to Regulate Cardiomyocyte Lipid Gene Expression and Oxidation. PPAR Research, 2015, 201625.

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Culturing Endothelial Cells for Research

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Bovine pulmonary artery endothelial cells (BPAEC) (cat. no. PB30205) were purchased from Genlantis (San Diego, CA). NIH/3T3 cells were obtained from ATCC (Manassas, VA). Those cells were cultured in DMEM (cat. no. VWRL0101–0500) supplemented with 10% fetal bovine serum (FBS) and 1X penicillin/streptomycin. Human lung microvascular endothelial cells (HMVEC-L) (cat. no. CC-2527) were obtained from Lonza (Walkersville, MD) and immortalized human cerebral microvascular endothelial cells (hCMEC/D3) (cat. no. SCC066) from Millipore Sigma (Temecula, CA). These cells were maintained in PromoCell Endothelial Cell Growth Medium MV. All cultures were maintained at 37°C in a humidified atmosphere of 5% CO₂-95% air. The non-immortalized cells were used at an early passage (<7).

Akhter M.S, & Barabutis N. (2021). Suppression of reactive oxygen species in endothelial cells by an antagonist of growth hormone releasing hormone. Journal of biochemical and molecular toxicology, 35(10), e22879.

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Murine Fam13a Promoter Activity Analysis

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All cell culture reagents were obtained from Fisher Scientific Inc. (Pittsburgh, PA). Insulin, dexamethasone, isobutylmethylxanthine (IBMX), puromycin were obtained from Sigma-Aldrich (St. Louis, MO). The 3T3-L1 (ATCC CL-173) and NIH/3T3 cells (ATCC CRL-1658) were cultured following ATCC instructions. pLightSwitch-Luc vector harboring predicted murine Fam13a promoter was obtained from Active Motif (Carlsbad, CA). pSV Sport PPARγ2 was obtained from Addgene (#8862). Promoter-driven luciferase activities were analyzed in NIH/3T3 cells by using LightSwitch™ Luciferase Assay System with or without PPARγ2 overexpression.

Tang J., Zhou H., Sahay K., Xu W., Yang J., Zhang W, & Chen W. (2018). Obesity-associated family with sequence similarity 13, member A (FAM13A) is dispensable for adipose development and insulin sensitivity. International Journal of Obesity (2005), 43(6), 1269-1280.

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Cell line origins and verification

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Embryonic stem cells (AN3-12 cells) are a clonal derivative of HMSc2, mouse embryonic stem cells derived in the laboratory (Elling et al. 2011 and 2017); NIH3T3 cells were purchased from ATCC, PlatinumE cells were purchased from Cell Biolabs, and LentiX cells were purchased from Clontech. A375 cells were obtained from Cancer Cell Line Encyclopedia. Hap1 cells were obtained from Haplogen and Hela cells from ATCC. All cell lines were confirmed to be mycoplasma negative regularly.

Chylinski K., Hubmann M., Hanna R.E., Yanchus C., Michlits G., Uijttewaal E.C., Doench J., Schramek D, & Elling U. (2019). CRISPR-Switch regulates sgRNA activity by Cre recombination for sequential editing of two loci. Nature Communications, 10, 5454.

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Establishment and Culture of Murine Embryonic Fibroblasts

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MEFs were established from approximately 14.5 dpc embryos as previously described³⁶ from Prdm16^+/− breeder pairs. Briefly, dissected embryo trunks were minced into 1–2mm fragments, resuspended in 3mL 0.25% trypsin/EDTA (Gibco, Carlsbad, CA) and passed 20–30 times through a 16ga needle. Cell suspensions were incubated at 37 °C for 1h with frequent agitation. Erythrocytes were lysed with ACK buffer, washed and cells were plated for 3h in 10% FBS/DMEM. Cells remaining in suspension were aspirated and adherent cells were cultured with fresh media. MEFs were passaged 1:3 every 3 days and cells between passage 2 and 5 were used for all experiments. 293 cells and NIH-3T3 cells were purchased from ATCC (Manassas, VA) and sub-cultured in 10% FBS/DMEM or 10% calf serum/DMEM, respectively. Wt and Mfn2^−/− MEFs were a kind gift from Dr. Eric Schon (Columbia University). All lines are tested yearly for Mycoplasma contamination and found negative.

Luchsinger L.L., de Almeida M.J., Corrigan D.J., Mumau M, & Snoeck H.W. (2016). Mitofusin 2 maintains hematopoietic stem cells with extensive lymphoid potential. Nature, 529(7587), 528-531.

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Cell Culture Maintenance and MEF Generation

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Cell lines were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific, 11965-118) supplemented with 10% bovine calf serum (ATCC, 30-2030) and 1X Penicillin–Streptomycin–Glutamine (Life Technologies, 10378016). Cultures were maintained at 37°C with 5% CO₂ and 95% humidity. MEFs were generated as previously described (Todaro and Green, 1963 (link)). NIH/3T3 cells (CRL-1658) and COS-7 cells (CRL-1651) were purchased from ATCC (Cat# CRL-1658). Plxna1^-/-;Plxna2^-/- MEFs were generated in the laboratory and authenticated using PCR. All cell lines were mycoplasma negative. Kif3a-/- NIH/3T3 Flp-In cells were obtained from Dr. Kristen Verhey (Engelke et al., 2019 (link)).

Pinskey J.M., Hoard T.M., Zhao X.F., Franks N.E., Frank Z.C., McMellen A.N., Giger R.J, & Allen B.L. (2022). Plexins promote Hedgehog signaling through their cytoplasmic GAP activity. eLife, 11, e74750.

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Cytotoxicity Evaluation of PEG-NaGdF4:Yb,Er NPs

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The cytotoxicity of the as-prepared PEG-NaGdF₄:Yb³⁺,Er³⁺ NPs was evaluated in vitro through an MTT assay using NIH3T3 cells (ATCC). The NIH3T3 cells were seeded at a density of 10⁴ cells/well in a 96-well plate and cultured in a minimum essential medium containing 10% fetal bovine serum and 1% penicillin–streptomycin at 37°C in a humidified atmosphere with 5% CO₂. The cells were exposed to different concentrations of the PEG-NaGdF₄:Yb³⁺,Er³⁺ NPs, ranging from 0.01 to 10 mM, for 24 h at 37°C. The cells were then washed with 9.6 mM PBS. A mixture of water-soluble tetrazolium salts, WST-8, and the culture medium was added to each well, and the cells were incubated for 30 min. The cell viability was evaluated by measuring the absorption of WST-8 at 450 nm in a microplate reader (Multiscan FC, Thermo Scientific, Massachusetts, US). The experiment was repeated five times.

Okubo K., Takeda R., Murayama S., Umezawa M., Kamimura M., Osada K., Aoki I, & Soga K. (2021). Size-controlled bimodal in vivo nanoprobes as near-infrared phosphors and positive contrast agents for magnetic resonance imaging. Science and Technology of Advanced Materials, 22(1), 160-172.

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Decellularization of Cell Culture Matrices

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bEnd.3 cells [American Type Culture Collection (ATCC CRL-2299)] were cultured in Dulbecco’s modified Eagle’s medium (DMEM):F12 (1:1) supplemented with 10% fetal bovine serum (FBS). NIH/3T3 cells (ATCC CRL-1658) were cultured in DMEM supplemented with 10% FBS. Cultures were decellularized to expose ECM using nonenzymatic cell removal techniques. Namely, versene treatment (three washes at 37°C for 10 min each) or three 10-min washes with 10 mM tris, 10 mM EDTA, and 1% Triton X-100 at 4°C, followed by 30-min incubation with deoxyribonuclease (0.2 mg/ml) in phosphate-buffered saline (PBS) with calcium and magnesium were used for decellularization (4 (link), 41 (link)). Following decellularization, all plates were stored for a maximum of 6 months at 4°C in PBS + 1% bovine serum albumin (BSA) until use. U87-MG (ATCC HTB-14) cells were grown in DMEM supplemented with 10% FBS and passaged using accutase.

Umlauf B.J., Clark P.A., Lajoie J.M., Georgieva J.V., Bremner S., Herrin B.R., Kuo J.S, & Shusta E.V. (2019). Identification of variable lymphocyte receptors that can target therapeutics to pathologically exposed brain extracellular matrix. Science Advances, 5(5), eaau4245.

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Generating GFP-Expressing shRNA Viral Vectors

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pLKO.1 shRNA expression plasmids (Sigma; Supplemental Table 4) were modified by cloning in the GFP cassette from pLKO.3G, a gift from Christophe Benoist & Diane Mathis (Addgene) (Primers listed in Supplemental Table 5). shRNA expression plasmids, RC-CMV-Rev1b, HDMHgpm2 (gag-pol), HDM-tat1b, HDM-VSV-G were transfected into HEK-293T cells (ATCC) using CalPhos™ Mammalian Transfection Kit (Takara Bio). Media was changed after 24hrs and virus was collected after 48hrs. Virus was titered using NIH/3T3 cells (ATCC).

Khokhar E.S., Borikar S., Eudy E., Stearns T., Young K, & Trowbridge J.J. (2020). Aging-Associated Decrease in the Histone Acetyltransferase KAT6B is Linked to Altered Hematopoietic Stem Cell Differentiation. Experimental hematology, 82, 43-52.e4.

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