Nih 3t3 mouse fibroblast

Manufactured by American Type Culture Collection

Sourced in United States

NIH 3T3 mouse fibroblasts are a cell line derived from mouse embryonic fibroblasts. The cells are widely used in cell biology research as a model system for studying various cellular processes.

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NIH-3T3 mouse embryo fibroblasts (2 citations)

38 protocols using nih 3t3 mouse fibroblast

Fibroblast Cell Culture Protocols

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All chemicals were purchased
from Fisher Scientific (Hampton, NH, USA) unless otherwise specified. p-coumaric acid (PCA) was purchased from TCI America Inc
(Portland, OR, USA). NIH 3T3 mouse fibroblasts and bacteria strains
were purchased from ATCC (Manassas, VA, USA).

Du C., Fikhman D.A., Persaud D, & Monroe M.B. (2023). Dual Burst and Sustained Release of p-Coumaric Acid from Shape Memory Polymer Foams for Polymicrobial Infection Prevention in Trauma-Related Hemorrhagic Wounds. ACS Applied Materials & Interfaces, 15(20), 24228-24243.

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Isolation and Characterization of Murine Fibroblast and Cardiomyocyte Populations

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NIH/3T3 mouse fibroblasts were purchased from ATCC. The WT19 fibroblasts were a gift from Dr. Anne Campbell (19 (link)). WT19 cells are mouse fibroblasts of the H-2^b haplotype obtained from C57Bl/6 mice and are immortalized by transformation with Simian Virus-40 T large antigen. Rat neonatal cardiac fibroblasts were obtained from the pre-plating step during standard isolation of neonatal cardiomyocytes (20 ). To insure absence of contamination with rat cardiomyocytes, the adherent neonatal cardiac fibroblasts were passaged in tissue culture-treated dishes two additional times, following the initial isolation. Mouse stem cell derived cardiac myocytes (mESC-CM) were purchased from LONZA (#XCAC-1010N). The mESC-CM were generated from the ES-D3 mouse embryonic stem cell line (ATCC, 129S4/Jae strain) by stable transduction with a bicistronic vector (21 (link)). This vector contains a cardiac-specific promoter driving the expression of both an antibiotic resistance gene and the EGFP cassette, ensuring selection of a high purity population of mESC-CM. The mESC-CM were maintained and differentiated according to the manufacturer’s instructions. Spontaneously beating mESC-CM layers were used within 3–14 days after cell plating. Where indicated, the cells were treated with 20 ng/mL recombinant murine interferon gamma (IFNγ) (PeproTech, NJ).

Karabekian Z., Idrees S., Ding H., Jamshidi A., Posnack N.G, & Sarvazyan N. (2015). Downregulation of beta-microglobulin to diminish T-lymphocyte lysis of non-syngeneic cell sources of engineered heart tissue constructs. Biomedical materials (Bristol, England), 10(3), 034101.

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HA Microrod Effects on 3T3 Fibroblasts

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NIH 3T3 mouse fibroblasts (ATCC, Manassas, Virginia) were cultured in the complete medium consisting of Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were harvested and cultured with HA microrods (100 mg/mL) at a high ratio (1:5) or a low ratio (1:20) of microrods to cells. Cell proliferation was assessed at 24, 48 and 72 hours after initial seeding using a CyQUANT assay. Experiments were done in triplicate. Genetic material was harvested and purified using the RNeasy Miniprep kit. RNA was converted into cDNA using the iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA). A Viia7 qPCR machine (Life Technologies, Carlsbad, CA) was used to measure relative expression levels of gene targets as compared to a housekeeping gene. Expression levels of all genes were evaluated using SYBR Green Mastermix (Life Technologies, Grand Island, NY) and custom-made DNA primers (Integrated DNA Technologies, Coralville, IA) in triplicate for three biological replicates (See Supplementary Table 1).

Le L.V., Mohindra P., Fang Q., Sievers R.E., Mkrtschjan M., Solis C., Safranek C.W., Russell B., Lee R.J, & Desai T.A. (2018). Injectable hyaluronic acid based microrods provide local micromechanical and biochemical cues to attenuate cardiac fibrosis after myocardial infarction. Biomaterials, 169, 11-21.

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Cultured Cell Lines and Fibroblasts

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293T cells (ATCC #CRL-3216) and NIH3T3 mouse fibroblasts (ATCC #CRL-1658) were obtained from ATCC and maintained with minor modifications according to the manufacture’s protocol. The primary mouse embryonic fibroblasts were established from Nestin-Cre^+/−;Cag-Cas9^+/+ mice and maintained in Dulbecco’s modified Eagle’s media (Gibco) supplemented with 10% fetal bovine serum (Gibco). Mouse neurospheres were generated and maintained as previously described [17 (link)].

Takadera M., Satomi K., Szulzewsky F., Cimino P.J., Holland E.C., Yamamoto T., Ichimura K, & Ozawa T. (2020). Phenotypic characterization with somatic genome editing and gene transfer reveals the diverse oncogenicity of ependymoma fusion genes. Acta Neuropathologica Communications, 8, 203.

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Culturing Mouse Fibroblasts and CHO Cells

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NIH 3T3 mouse fibroblasts (ATCC, Manassas, VA) were grown in Dulbecco’s Modified Eagle’s Medium (DMEM; Life Technologies, Grand Island, NY) with 10% bovine calf serum (Hyclone, Logan, UT) and antibiotic/antimycotic cocktail (Corning Life Sciences, Oneonta, NY). Wild-type CHO-K1 (ATCC) and mutant CHO-677 cells (a gift from Dr. Jeffrey Esko, UCSD) [22 (link)] were maintained in DMEM containing 10% FetalClone Serum (Hyclone) and antibiotic/antimycotic cocktail. All cell lines used were tested and found to be free of mycoplasma contamination.

Raitman I., Huang M.L., Williams S.A., Friedman B., Godula K, & Schwarzbauer J.E. (2017). Heparin-Fibronectin Interactions in the Development of Extracellular Matrix Insolubility. Matrix biology : journal of the International Society for Matrix Biology, 67, 107-122.

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Cultivation and Characterization of HCMV Strains

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Primary human foreskin fibroblasts (HFFs) and adipose-derived adult mesenchymal stem cells (ASCs) were cultivated as previously described [18 (link),19 (link)].
The wild-type HCMV strain, Hi91, was isolated from the urine of an AIDS patient with HCMV retinitis, as described previously [20 (link)]. HCMV strains Davis and Towne were received from the American Type Culture Collection (ATCC) (Manassas, VA, USA). Virus stocks were prepared in HFFs maintained in minimal essential medium (MEM), supplemented with 4% fetal calf serum (FCS). The U1, U59, and U75 are patient isolates, which were isolated as previously described [20 (link),21 (link)]. Virus stocks were prepared in HFFs maintained in MEM supplemented with 4% FCS.
Murine cytomegalovirus (Smith strain, catalogue number VR-1399) was obtained from ATCC, and virus stocks were prepared in NIH/3T3 mouse fibroblasts (ATCC) maintained in MEM supplemented with 4% FCS.
DNA isolation, amplification, and sequencing were performed as previously described [21 (link)], using established primers [22 (link)].

Vogel J.U., Schmidt S., Schmidt D., Rothweiler F., Koch B., Baer P., Rabenau H., Michel D., Stamminger T., Michaelis M, & Cinatl J. (2019). The Thrombopoietin Receptor Agonist Eltrombopag Inhibits Human Cytomegalovirus Replication Via Iron Chelation. Cells, 9(1), 31.

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Fibroblast Cell Culture and Treatment

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NIH-3T3 mouse fibroblasts were purchased from ATCC (USA). The cells were inoculated into a cell culture dish and cultured in a medium composed of 89% DMEM/F12 (Hyclone, USA) supplemented with 10% fetal bovine serum (Sijiqing, China) and 1% penicillin/streptomycin (Procell, China). Cell culture was conducted in an incubator containing 5% CO₂ at 37°C.
The cultured cells were categorized into four groups: CON, AngII, SXSM, and SC79. CON was used as a blank control group without any intervention measurements. The cells in the other groups were treated with AngII (Sigma-Aldrich, USA) for 48 h after culturing for 12 h, while the SXSM group was treated with SXSM, and the SC79 group was treated with SXSM in combination with the AKT phosphorylation agonist SC79 (Topscience, China). The concentration of AngII was 0.1 μM, and those of SXSM and SC79 were 1 mL/L and 8 μg/mL, respectively. The optimal concentration of SXSM was obtained by cell viability testing.

Chen C., Zhang H., Hou S., Liu Y., Ren L., Hao M., Chen K., Li L., Cai X., Deng Z., Liu Z, & Hou P. (2022). Shenxian-Shengmai Oral Liquid Evoke Autophagy of Fibroblast to Attenuate Sinoatrial Node Fibrosis in Sick Sinus Syndrome Mice via the AKT/mTOR Pathway. Evidence-based Complementary and Alternative Medicine : eCAM, 2022, 5219277.

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Pharmacological Inhibition of 3T3 Fibroblasts

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NIH/3T3 mouse fibroblasts (ATCC, Manassas, VA, USA) were cultured in DMEM medium (Gibco, New York, NY, USA) supplemented with 10% fetal calf serum (Gibco) and 1% penicillin–streptomycin solution (Gibco), pH 7.4 at 37 °C, 5% CO2, 95% humidity. Medium was changed every 2 days. When reaching 80–90% confluence, cells were pre-treated with various pharmacological inhibitors for different durations including n-MPG (in distilled water, final concentrations 300 µM, Sigma-Aldrich, St. Louis, MN, USA), PDTC (in distilled water, 2 mM, Sigma-Aldrich), DPI (in DMSO, 10 µM, Calbiochem, San Diego, CA, USA), Apo (in DMSO, 30 µM, Calbiochem), and Cyto D (in DMSO, 2 nM, Sigma-Aldrich) for 30 min, zVAD-fmk (in DMSO, 50 µM, BioVision, Milpitas, CA, USA) for 1 h, and Nec-1 (in DMSO, 10 µM, BioVision) for 2 h. After replacing with new medium, cells were cultured with NSP and collected at indicated time points for further analyses. NSP were dispersed in PBS buffer with sonication for 10 min prior to treatment.

Huang J.T., Chang L.C., Cheng C.S., Lin J.J., Huang S.Y, & Chen S.E. (2020). Cytotoxicity Produced by Silicate Nanoplatelets: Study of Cell Death Mechanisms. Toxins, 12(10), 623.

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Cell culture conditions for cancer and fibroblast cells

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4T1 mouse breast cancer cells (ATCC, Manassas, VA) were grown in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), and LS174T human colon cancer cells (ATCC, Manassas, VA) in Eagle’s Minimal Essential Medium (EMEM) containing 10% FBS. NIH/3T3 mouse fibroblasts (ATCC, Manassas, VA) and J774A.1 macrophages (ATCC, Manassas, VA) were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% calf bovine serum. All the media contained 100 units/mL of penicillin and 100 μg/mL of streptomycin. Cells were incubated at 37 °C in a humidified 5% CO₂ atmosphere.

Park J., Pei Y., Hyun H., Castanares M.A., Collins D.S, & Yeo Y. (2017). Small molecule delivery to solid tumors with chitosan-coated PLGA particles: A lesson learned from comparative imaging. Journal of controlled release : official journal of the Controlled Release Society, 268, 407-415.

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TGF-β1 and Decorin Regulation of Gene Expression

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NIH 3T3 mouse fibroblasts (ATCC, Manassas, Virginia) were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal calf serum and 1% penicillin/streptomycin. Cells were seeded into 24-well plates and then media was added that either contained 10 ng/mL TGF-β1 (PeproTech 100-21) or 10 ng/mL TGF-β1 plus 10 μg/mL decorin. Genetic material was harvested and purified using the RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA was converted into cDNA using the iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA) and a Viia7 qPCR machine (Life Technologies, Carlsbad, CA) was used to measure relative expression levels of gene targets compared to the housekeeping gene 60 S ribosomal protein L19. Expression levels of all genes were evaluated using the Fast SYBR Green Mastermix (Life Technologies, Grand Island, NY) and custom DNA primers (Integrated DNA Technologies, Coralville, IA) in triplicate for three biological replicates (Table S1).

Mohindra P., Zhong J.X., Fang Q., Cuylear D.L., Huynh C., Qiu H., Gao D., Kharbikar B.N., Huang X., Springer M.L., Lee R.J, & Desai T.A. (2023). Local decorin delivery via hyaluronic acid microrods improves cardiac performance, ventricular remodeling after myocardial infarction. NPJ Regenerative Medicine, 8, 60.

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