Neuro 2a

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The Neuro-2a is a mouse neuroblastoma cell line commonly used in cell biology and neuroscience research. It is a well-established model for studying neural cell function and differentiation.

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Neuro-2a cells (4 citations)

24 protocols using neuro 2a

Culturing Neuro-2a and PC-12 Cells

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The Neuron-2a cells were obtained from the American Type Culture Collection (ATCC) (Rockville, MD, USA). The differentiated PC-12 cell line was purchased from Kunming Cell Bank of the Chinese Academy of Science (Kunming, Yunnan, China). Both the Neuro-2a and differentiated PC-12 cells were cultured with DMEM (Gibco, Grand Island, NE, USA) containing 10% fetal bovine serum supplied with 50 U/mL penicillin and 50 μg/mL streptomycin (Invitrogen, Scotland, UK). The FBS was replaced with exosome-depleted FBS to culture the cells before they were subjected to the further isolation of the exosomes [63 (link)]. All of the cells were cultured in a 5% humidified CO₂ incubator at 37 °C.

Tang B., Zeng W., Song L.L., Wang H.M., Qu L.Q., Lo H.H., Yu L., Wu A.G., Wong V.K, & Law B.Y. (2022). Extracellular Vesicle Delivery of Neferine for the Attenuation of Neurodegenerative Disease Proteins and Motor Deficit in an Alzheimer’s Disease Mouse Model. Pharmaceuticals, 15(1), 83.

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Culturing Mouse Cancer Cell Lines

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Neuro2A
(mouse neuroblastoma)
and GL-261 (mouse glioblastoma) lines were obtained from the American
Type Culture Collection (ATCC). Neuro2A and GL-261 cells were maintained
in Dulbecco’s modified Eagle’s medium (DMEM) supplemented
with 10% FBS (Gibco), penicillin/streptomycin (1×), Non-Essential
Amino Acids Solution (1×), and sodium pyruvate (1×) at 37
°C in a humidified atmosphere supplemented with 5% CO₂.

Reddy R.G., Surineni G., Bhattacharya D., Marvadi S.K., Sagar A., Kalle A.M., Kumar A., Kantevari S, & Chakravarty S. (2019). Crafting Carbazole-Based Vorinostat and Tubastatin-A-like Histone Deacetylase (HDAC) Inhibitors with Potent in Vitro and in Vivo Neuroactive Functions. ACS Omega, 4(17), 17279-17294.

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Cell Culture and Transfection Protocol

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HEK 293 T (CRL-11268), HeLa (CCL-2), MCF7 (HTB-22), HT1080 (CCL-121), and Neuro-2a (CCL-131) cells were purchased from American Type Culture Collection (ATCC). HEK 293 T, HeLa, and Neuro-2a cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% fetal bovine serum (FBS) (Corning) at 37°C with 10% or 5% CO₂ under standard humidity. MCF7 and HT1080 cells were cultured in Eagle’s minimum essential medium with 10% FBS (Corning) at 37°C with 10% CO₂ under standard humidity. The cells were transfected using Lipofectamine LTX (Invitrogen) according to the manufacturer’s recommendations.

Yu J., Shin J., Yu J., Kim J., Yu D, & Heo W.D. (2024). Programmable RNA base editing with photoactivatable CRISPR-Cas13. Nature Communications, 15, 673.

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Culturing Neuroblastoma Cell Lines

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Neuro2a (mouse neuroblastoma)
and IMR32 (human neuroblastoma) cell lines were procured from the
American Type Culture Collection. These cell lines (Neuro2a and IMR32)
were maintained in complete media containing Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum
(FBS) (Gibco), penicillin/streptomycin (1%), sodium pyruvate (1×),
and nonessential amino acids solution (1×), at 37 °C in
a humidified atmosphere supplemented with 5% CO₂. For differentiation
experiments, cells were grown in culture media with reduced serum
(DMEM + 1% FBS).

Reddy R.G., Dachavaram S.S., Reddy B.R., Kalyankar K.B., Rajan W.D., Kootar S., Kumar A., Das S, & Chakravarty S. (2018). Fellutamide B Synthetic Path Intermediates with in Vitro Neuroactive Function Shows Mood-Elevating Effect in Stress-Induced Zebrafish Model. ACS Omega, 3(9), 10534-10544.

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Cell Culture Protocol for Common Cell Lines

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HEK‐293 (human, female, RRID: CVCL_0045), U937 (human, male, RRID: CVCL_0007) and Neuro‐2A (mouse, male, RRID: CVCL_0470) cells were purchased from ATCC (Cat# CRL‐1573, CRL‐1593, CCL‐131, respectively). HEK‐293 and Neuro‐2A cells were cultured in DMEM (Gibco), supplemented with 10% fetal bovine serum (Gibco), 100 U/ml penicillin, and 100 μg/ml streptomycin. U937 cells were grown in RPMI‐1640 medium (Biological Industries) supplemented with 10% fetal bovine serum (Gibco), 100 U/ml penicillin, and 100 μg/ml streptomycin. All cells were incubated at 37°C (Farin et al, 2011 (link)) and 5% CO₂. The cell lines used were not authenticated.

Doron‐Mandel E., Koppel I., Abraham O., Rishal I., Smith T.P., Buchanan C.N., Sahoo P.K., Kadlec J., Oses‐Prieto J.A., Kawaguchi R., Alber S., Zahavi E.E., Di Matteo P., Di Pizio A., Song D., Okladnikov N., Gordon D., Ben‐Dor S., Haffner‐Krausz R., Coppola G., Burlingame A.L., Jungwirth P., Twiss J.L, & Fainzilber M. (2021). The glycine arginine‐rich domain of the RNA‐binding protein nucleolin regulates its subcellular localization. The EMBO Journal, 40(20), e107158.

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Knockdown of ANT1 and ANT2 in Neuro-2a Cells

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The cell line Neuro-2a (CCL-131) was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), maintained in the complete medium of high glucose DMEM (SH30243.01, HyClone, Los Angeles, USA) supplemented with 1% antibiotics (GIBCO, USA), 10% fetal bovine serum (FBS) (10270-106, GIBCO, Massachusetts, USA) and 0.5% GlutaMAX (35050-061, GIBCO, Massachusetts, USA) in the incubator of 37 • C and 5% CO 2 .
To reduce ANT1 and ANT2 in the cell line of Neuro-2a, we designed a shRNA targeted to ANT1 with adenovirus as vector (pADV-U6-shRNA (Slc25a4)-CMV-EGFP). The target sequence was: GCAGTTCTGGCGC-TACTTT, which has a high degree of homology with ANT2 for one base pair difference. Both ANT1 and ANT2 were effectively knocked down in
the Neuro-2a by this shRNA. The stock solution of virus was 5.53 × 10 10 PFU (plaque forming unit)/ml in titer. The MOI (Multiplicity of infection) was 100 IFU (infect formation unit)/cell in the experiment. For the ANT1 knock down singly, the target sequence was: GCTGGTGTCCTATCCGTTT. For the ANT2 knock down singly, the target sequence was: CTTGGTGACTGCCTGGTTA. The MOI used in knocking down singly ANT1 or ANT2 was also 100 IFU. After 48 h of virus transfection, the cells were used in the experiments.

Xu Y., Peng S., Cao X., Qian S., Shen S., Luo J., Zhang X., Sun H., Shen W., Jia W., & Ye J. (2021). High doses of butyrate induce a reversible body temperature drop through transient proton leak in mitochondria of brain neurons. Life Sciences, 278, 119614.

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Cell Cultivation Protocols for Assays

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The neuroblastoma cells (Neuro-2A), monocytes (THP-1), liver hepatocellular carcinoma cell (HepG2), human embryonic kidney cells (HEK), colon carcinoma cells (HCT-116) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). The human bronchial epithelial cells (16HBE) was a gift form Dr. D. C. Gruenert (University of California, San Francisco) [74 (link)]. HepG2, 16HBE, HEK, and HCT116 cells were cultured in Dulbecco’s Modified Eagle Medium (D MEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA). Neuro-2A and THP-1 were cultured in minimum Eagle’s medium (MEM, Gibco, USA) and Roswell Park MEMorial Institute-1640 (RPMI-1640, Gibco, USA), respectively, supplemented with 10% FBS. Cells were incubated at 37 °C in a humidified chamber with 5% CO₂.

Li D., Chen S., Li Q., Chen L., Zhang H., Li H., Yu D., Zhang R., Niu Y., Lu S., Ye L., Zeng X., Dong G., Chen R., Aschner M., Zheng Y, & Chen W. (2020). Caloric restriction attenuates C57BL/6 J mouse lung injury and extra-pulmonary toxicity induced by real ambient particulate matter exposure. Particle and Fibre Toxicology, 17, 22.

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Cell Culture Conditions and Validation

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A549 cells and Neuro-2a cells were obtained from American Type Culture Collection (ATCC). A549 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) (Gibco, 10270-106) and antibiotics (100μg/ml penicillin and 50μg/ml streptomycin sulfate) (Gibco, 15140122). Neuro-2a cells were maintained in modified Eagle's medium (MEM) with 10% FBS, antibiotics (100μg/ml penicillin and 50μg/ml streptomycin sulfate) and 1% NEAA (Gibco, 11140050). HUVECs were bought from ScienCell Research Laboratories (ScienCell, 8000) and cultured in EGM-2 medium (Lonza, CC-3162). All cell lines were genotyped to confirm their identity at Genewiz. Cells were incubated at 37℃ with 5% CO2, and tested routinely for Mycoplasma contamination.

Luan Y., Tang N., Yang J., Chen C., Liu S., Cheng C., Wang Y., Guo Y., Wang H., Xiang M., Ju R., & Xie Z. (2021). Decoding regulatory specificity of human ribosomal proteins.

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Cell Culture Protocols for Multiple Cell Lines

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PC-12, Neuro2A, HEK 293, HeLa, and MDA-MB-231 cell lines were obtained from American Type Culture Collection (Rockville, MD, USA). HEK 293, HeLa, Neuro2A, MDA-MB-231 cells were grown in DMEM medium, supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA, USA) and penicillin/streptomycin (Invitrogen). PC-12 cells were grown in RPMI medium supplemented with 10% horse serum and 5% FBS. We previously generated PC-12 Tet-on/off cell lines stably expressing NRP/B.22 (link) The Tet-on/off PC12 clones were maintained as described.

Choi J., Yang E.S., Cha K., Whang J., Choi W.J., Avraham S, & Kim T.A. (2014). The Nuclear Matrix Protein, NRP/B, Acts as a Transcriptional Repressor of E2F-mediated Transcriptional Activity. Journal of Cancer Prevention, 19(3), 187-198.

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Cultured Cell Lines and Primary Neuron Isolation

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COS-7 and Neuro-2a cells (ATCC, Manassas, VA, USA) were cultured in DMEM (Invitrogen) supplemented with 10% heat-inactivated FBS plus antibiotics in an incubator at 37°C with 5% CO₂. Primary cortical neuron cultures were prepared from embryonic day 18 (E18) SD rats. E18 timed pregnant rats were euthanized by carbon dioxide inhalation and rapid decapitation. The E18 embryos subsequently removed and sacrificed by decapitation. Cortices were then dissociated in trypsin and plated on poly-d-lysine–coated culture plates. Cells were cultured in Neurobasal medium supplemented with 2% B27 and antibiotics. All animal experiments were conducted in accordance with the protocol number 2009056 approved by the Animal Care Committee of the Hong Kong University of Science and Technology. To examine the effects of blocking the nuclear export and inhibition of Cdk5 activity, the cells were treated with leptomycin B (LMB, 5–10 ng/mL; Sigma, St. Louis, MO, USA) or roscovitine (10–25 µM; Sigma), respectively, for 4 or 8 h before analysis.
COS-7 and Neuro-2a cells were transfected with Lipofectamine Plus reagents (Invitrogen). Meanwhile, cortical neurons were transfected with the nucleofector transfection kit (Amaxa) or a calcium phosphate transfection assay.

Zhao X.S., Fu W.Y., Chien W.W., Li Z., Fu A.K, & Ip N.Y. (2014). p35 Regulates the CRM1-Dependent Nucleocytoplasmic Shuttling of Nuclear Hormone Receptor Coregulator-Interacting Factor 1 (NIF-1). PLoS ONE, 9(10), e110584.

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