Nih3t3

Manufactured by American Type Culture Collection

Sourced in United States, China, Germany, Japan, Israel, Italy

NIH3T3 is a fibroblast cell line derived from the Swiss albino mouse embryo. It is a commonly used model for studying cell growth, differentiation, and signaling pathways.

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NIH3T3 cells (243 citations) NIH3T3 mouse embryonic fibroblasts (9 citations) NIH3T3 mouse fibroblast cell line (5 citations) NIH3T3-E1 fibroblasts (3 citations) NIH3T3 mouse embryonic fibroblast cells (3 citations) Murine NIH3T3 fibroblasts (3 citations) Mouse NIH3T3 cells (2 citations) NIH3T3 MEFs (2 citations) NIH3T3 fibroblast cell lines (ATCC® CRL-1658) (2 citations)

543 protocols using nih3t3

Cell Line Cultivation and Characterization

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293T (human embryonic kidney transformed with SV40 T antigen, ATCC^® CRL-3216™), A375 (human melanoma, ATCC^® CRL-1619™), CT 26 (mouse colon carcinoma, ATCC^® CRL-2638™), and NIH/3T3 (mouse embryonic fibroblasts, ATCC^®, CRL-1658™) cell lines were obtained from ATCC^® (Manassas, VA, USA).
293T, A375, and NIH/3T3 cell lines were cultured in DMEM/F12 medium (1:1) supplemented with 10% FBS and antibiotic-antimycotic solution in a 5% CO₂ incubator at 37 °C under 95% humidity.
The CT 26 cell line was cultured in RPMI-1640 medium supplemented with 12.5% FBS and antibiotic-antimycotic solution in a 5% CO₂ incubator at 37 °C under 95% humidity.
All the cell lines were subcultured every 3–4 days using trypsin-EDTA. For mRNA content characterization and transfection experiments, cells were collected with trypsin-EDTA, stained with trypan blue stain (Invitrogen), and counted using the Countess II FL Automated Cell Counter (Invitrogen).

Kuzmich A., Rakitina O., Didych D., Potapov V., Zinovyeva M., Alekseenko I, & Sverdlov E. (2020). Novel Histone-Based DNA Carrier Targeting Cancer-Associated Fibroblasts. Polymers, 12(8), 1695.

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Cell Culture Protocols for Fibroblast and Lung Cell Lines

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Mouse fibroblast cells (NIH3T3, purchased from ATCC, passage number 32-37), (CAGA)₁₂-Lux reporter stably transfected NIH 3T3 cells (CAGA-NIH3T3, passage number 30-35) and mouse lung fibroblast cells (Mlg, purchased from ATCC, passage number 25-30) were grown in DMEM (KeyGEN BioTECH, Nan Jing, China) supplemented with 10% fetal bovine serum (FBS, ExCell Bio, Shanghai, China). A549 cells (purchased from ATCC, passage number 27-29) were grown in RPMI-1640 (KeyGEN BioTECH, Nan Jing, China) supplemented with 10% FBS. Cells were maintained at 37°C with 5% CO₂ in a humidified atmosphere.

Li X., Bi Z., Liu S., Gao S., Cui Y., Huang K., Huang M., Mao J., Li L., Gao J., Sun T., Zhou H, & Yang C. (2019). Antifibrotic Mechanism of Cinobufagin in Bleomycin-Induced Pulmonary Fibrosis in Mice. Frontiers in Pharmacology, 10, 1021.

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Cell Culture Protocol for HepG2 and NIH 3T3

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Two different cell lines, HepG2 and NIH 3T3 (both from ATCC, Manassas, VA, United States) were cultured to ∼90% confluence after 21–25 h of growth under standard conditions (5% CO₂/95% O₂ at 37°C): Seeding densities for HepG2 and NIH 3T3 were 6.97 × 10⁴ and 6.67 × 10⁴ cells/cm², respectively. HepG2 cells were cultured in Eagle’s Minimal Essential Medium supplemented with 10% (v/v) fetal bovine serum (FBS), 1% (v/v) of non-essential amino acid (NEAA) mixture, and 1 mM sodium pyruvate. NIH 3T3 cells were cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10% (v/v) newborn calf serum (NCS) (Gibco, Paisley, United Kingdom). All culture media were supplemented with penicillin (100 IU/mL), streptomycin (100 μg/mL), and L-glutamine (2 mM). All cell media and supplements were obtained from Sigma-Aldrich (St. Louis, MO, United States), except serum (Gibco, Paisley, United Kingdom). The 96-well plates were from Corning Costar (Sigma-Aldrich, Brøndby, Denmark).

Molchanova N., Wang H., Hansen P.R., Høiby N., Nielsen H.M, & Franzyk H. (2019). Antimicrobial Activity of α-Peptide/β-Peptoid Lysine-Based Peptidomimetics Against Colistin-Resistant Pseudomonas aeruginosa Isolated From Cystic Fibrosis Patients. Frontiers in Microbiology, 10, 275.

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Characterization of IFNAR1-deficient cells

Cited 1 time

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MRC-5 fibroblasts (ATCC CCL-171), NIH3T3 (ATCC CRL-1658), NIH3T3:ISRE-luciferase21 (link), primary and crisis immortalized IFNAR1-deficient cells (generated from IFNAR1-deficient MEF41 (link)), crisis immortalized C57BL/6 cells42 (link), Vero (ATCC CCL-81), A549 (ATCC CCL-185), CV-1 (ATCC CCL-70) and MDCK cells were grown in DMEM supplemented with 10% (v/v) FCS, streptomycin, penicillin and 2 mM glutamine.
Mouse IFN-α (#12100-1) was purchased from PBL Biomedical Laboratories, New Jersey, USA. MLN4924 was purchased from Active Biochem (#A-1139).

Le-Trilling V.T., Megger D.A., Katschinski B., Landsberg C.D., Rückborn M.U., Tao S., Krawczyk A., Bayer W., Drexler I., Tenbusch M., Sitek B, & Trilling M. (2016). Broad and potent antiviral activity of the NAE inhibitor MLN4924. Scientific Reports, 6, 19977.

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Cell Lines and Antibodies Used

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The following cells were used in experiments as indicated: U2OS (American Type Culture Collection ID HTB-96); NIH-3T3 (American Type Culture Collection ID CRL-1658) were received from Dr. Craig Blackstone’s laboratory (National Institute of Neurological Disorders and Stroke, Bethesda, MD); NIH-3T3 ATL1/2/3 TKO cells (E21 cell line) were engineered by the Blackstone laboratory using CRISPR editing, resulting in each atl allele having small insertion or deletion mutations (1–7 nt) in exon regions (described in Zhao et al., 2016 (link)). The following antibodies were used as indicated in subsequent sections: anti-c-Myc monoclonal antibody (mouse) 9E10 (Thermo Fisher Scientific; cat #MA1-980); anti-calnexin polyclonal (rabbit; Abcam; cat #ab22595); EZview Red anti-c-Myc (rabbit) affinity gel (Sigma-Aldrich; cat #E6654); Goat anti-mouse IgG cross-adsorbed secondary antibody, Alexa Fluor 488 (Thermo Fisher Scientific; cat #A-11017); goat anti-mouse IgG (heavy and light chains) HRP-conjugated secondary antibody (Thermo Fisher Scientific; cat #62-6520); and goat anti-rabbit IgG (heavy and light chains) HRP-conjugated secondary antibody (Thermo Fisher Scientific; cat #65-6120).

Kelly C.M., Byrnes L.J., Neela N., Sondermann H, & O’Donnell J.P. (2021). The hypervariable region of atlastin-1 is a site for intrinsic and extrinsic regulation. The Journal of Cell Biology, 220(11), e202104128.

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Cell Culture and Transfection Protocols

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The NSCLC cell lines A549 and H1299, mouse Lewis lung cancer cell line LLC, mouse embryonic fibroblast cell line NIH 3T3, mouse melanoma cell line B16, mouse leukemic monocyte macrophage cell line RAW 264.7, and human bronchial epithelial cell line BEAS-2B were purchased from the ATCC (Philadelphia, PA, USA). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) or RPMI 1640 (Invitrogen, Carlsbad, CA, USA), supplemented with 10% (v/v) fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) and 1% penicillin–streptomycin (Invitrogen, Carlsbad, CA, USA). All cells were cultured in a humidified CO₂ incubator at 37 °C. Plasmids were introduced into cells via polyethylenimine (PEI)-mediated transfection as described previously^{24 (link)}. Briefly, cells were seeded at a density of 2 × 10⁶ cells/10 cm dish (Corning, Lowell, MA, USA) and were transfected with PEI-complexed plasmids (at a ratio of 2:1, w/w) in serum-free DMEM. Two hours after addition of the DNA, the medium was replaced with fresh complete medium, and transfected cells were continuously cultured until harvested for analysis.

Jiang W., Jin G., Cai F., Chen X., Cao N., Zhang X., Liu J., Chen F., Wang F., Dong W., Zhuang H, & Hua Z.C. (2019). Extracellular signal-regulated kinase 5 increases radioresistance of lung cancer cells by enhancing the DNA damage response. Experimental & Molecular Medicine, 51(2), 1-20.

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Mouse Cell Lines for Cancer Research

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The mouse prostate epithelial cell line MPR31.4 was a kind gift of TC Thompson, Scott (Department of Urology, Baylor College of Medicine, Houston, USA) (Thompson et al., 1993 (link); Shaker et al., 2000 (link)). The mouse breast cancer cell line Py8119, the skin and embryonic fibroblasts cell lines L929, NIH-3T3 were purchased from ATCC (Manassas, VA, USA).

Steer A., Cordes N., Jendrossek V, & Klein D. (2019). Impact of Cancer-Associated Fibroblast on the Radiation-Response of Solid Xenograft Tumors. Frontiers in Molecular Biosciences, 6, 70.

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Culturing Fibroblast Cell Lines

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LEADING LIGHT Wnt Reporter cells (Enzo Life Sciences) and NIH/3T3 (ATCC CRL-1658, ATCC, RRID:CVCL_0594) cells, both fibroblast cell lines, were cultured in high-glucose DMEM (Gibco), supplemented with 4-mM l-alanyl-l-glutamine dipeptide (Gibco), 10% (w/v) FBS (PAN-Biotech) and 100-U·ml⁻¹ penicillin and streptomycin (Gibco). For all experiments using NIH/3T3 and LEADING LIGHT Wnt Reporter cells, well plates were coated with poly-l-lysine (Sigma-Aldrich). MLg (ATCC CCL206, ATCC, RRID: CVCL_0437) cells were cultured in DMEM/Ham’s F12 media (Gibco) supplemented with 10% (w/v) FBS and 100-U·ml⁻¹ penicillin and streptomycin. Cells were grown at 37°C with 5% CO₂ in humidified conditions.

Costa R., Wagner D.E., Doryab A., De Santis M.M., Schorpp K., Rothenaigner I., Lehmann M., Baarsma H.A., Liu X., Schmid O., Campillos M., Yildirim A.Ö., Hadian K, & Königshoff M. (2021). A drug screen with approved compounds identifies amlexanox as a novel Wnt/β-catenin activator inducing lung epithelial organoid formation. British journal of pharmacology, 178(19), 4026-4041.

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Breast Cancer Cell Conditioning for Exosome Study

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Human monocyte cell line THP-1, breast cancer cell lines MDA-MB-231, T47D and normal breast cell line MCF10A were procured from NCCS, India. All cell lines were routinely cultured in R10 media (RPMI-1640, 10% FBS, penicillin (100 I.U./mL), streptomycin (100 μg/mL), L-glutamine (2 mM), sodium pyruvate (0.5 mM)) (Thermo). For MCF10A, medium was additionally supplemented with insulin (10 μg/mL), cholera toxin (100 ng/mL) and EGF (20 ng/mL) (Peprotech).
Murine fibroblast cell line NIH/3T3, procured from ATCC, USA, and breast cancer cell line Brpkp110 were routinely maintained in R10 media, or cultured in 10% exosome-depleted-FBS-containing RPMI.
For conditioned media collection, breast cancer cell lines MDA-MB-231 and T47D were grown to 60-70% confluency before replacing the medium with fresh R10. Both cell lines were then cultured for another 48 hr and media were collected. Further, conditioned media were filtered using 0.45 μ filter (Sartorius) to remove cell debris.

Biswas S., Mandal G., Chowdhury S.R., Purohit S., Payne K.K., Anadon C., Gupta A., Swanson P., Yu X., Conejo-Garcia J.R, & Bhattacharyya A. (2019). EXOSOMES PRODUCED BY MESENCHYMAL STEM CELLS DRIVE DIFFERENTIATION OF MYELOID CELLS INTO IMMUNOSUPPRESSIVE M2-POLARIZED MACROPHAGES IN BREAST CANCER. Journal of immunology (Baltimore, Md. : 1950), 203(12), 3447-3460.

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Cell Culture and Metabolic Labeling

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U2OS cells (American Tissue and Cell Collection, HTB-96) were a kind gift of Dr. John Hogenesh (University of Pennsylvania) NIH-3T3 (CRL-1658), MDCK (CCL-34) and 293T cells (CRL-3216) were directly obtained from ATCC. All cell lines were cultured in 10% Dulbecco’s modified Eagle medium supplemented to 10% decomplemented fetal bovine serum at 37°C, 5% CO₂, 21% O₂ and 100% humidity unless otherwise indicated. For aminoacid depletion experiments, dialyzed FBS was used and L-glutamine was supplemented as indicated. Cell lines were maintained and passaged according to ATCC recommended procedures. Pharmacological agents was as follows: Purvalanol-A (Sigma P4484), Z-VAD-FMK (EMD Millipore 627610), actinomycin D (Sigma A9415), cycloheximide (Sigma C7698), NAC (Sigma A9165), Trolox (Santa Cruz Biotech sc-200810) and CCCP (Sigma C2759) were added to cell cultures 12 h after cell seeding at the concentrations indicated and maintained until analysis, changing the medium every 24 hours. Ethynyl-uridine (Life Technologies E10345) and homopropargyl-glycine (Life Technologies C10186) were added at 5 and 50 μM respectively, two hours prior to incorporation analysis using the Click-iT alkyne detection kit (Life Technologies C10330).

Penzo-Méndez A.I., Chen Y.J., Li J., Witze E.S, & Stanger B.Z. (2015). Spontaneous Cell Competition in Immortalized Mammalian Cell Lines. PLoS ONE, 10(7), e0132437.

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