N2 medium

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N2 medium is a defined, serum-free cell culture supplement that supports the growth and maintenance of various cell types, including neurons and neural stem cells. It is designed to provide a controlled and chemically defined environment for optimal cell proliferation and differentiation.

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N2 medium supplement (3 citations) DMEM-F12/glutamax medium with N2 supplement (2 citations) DMEM-N2 medium (2 citations)

11 protocols using n2 medium

Optimizing Neural Stem Cell Culture Media

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NB medium: neurobasal medium (Gibco, Waltham, MA, USA, 21103-049) supplemented with 2% B27 with vitamin A (Gibco, Waltham, MA, USA, 17504-044), 18 mM Hepes (Gibco, Waltham, MA, USA, 15630-056), 0.25× Glutamax (Gibco, Waltham, MA, USA, 35050-038), 1% Pen/Strep (Sigma-Aldrich, Saint Louis, MO, USA, P0781).
N2 medium: Dulbecco’s modified Eagle medium/nutrient mixture F12 medium (DMEM/F12) (Life Technologies, Waltham, MA, USA, #21331-046) supplemented with 1% N2 (Thermo Fisher Scientific, Waltham, MA, USA, #17502-048), 1× NEAA (Thermo Fisher Scientific, Waltham, MA, USA, #11140-035), 2 mM L-glutamine (Thermo Fisher Scientific, Waltham, MA, USA, #25030-024), 2 μg/mL heparin (Sigma-Aldrich, Saint Louis, MO, USA, #H3393-50KU), 1% Pen/Strep.
N2B27—vit A: 1:1 DMEM/F12 with neurobasal medium supplemented with 0.5% N2, 1% B27 without vitamin A (Thermo Fisher Scientific, Waltham, MA, USA, #12587-010), 2.5 μg/mL insulin (Sigma-Aldrich, Saint Louis, MO, USA, #I9278), 0.5% L-glutamine, 0.5× NEAA, 50 μM beta-mercaptoethanol (Thermo Fisher Scientific, Waltham, MA, USA, #21985023), 1% Pen/Strep
N2B27 + vit A: 1:1 DMEM/F12 with neurobasal medium supplemented with 0.5% N2, 1% B27 with vitamin A, 0.25 mg/mL insulin, 0.5% L-glutamine, 0.5× NEAA, 50 μM beta-mercaptoethanol, 1% Pen/Strep

Dooves S., van Velthoven A.J., Suciati L.G, & Heine V.M. (2021). Neuron–Glia Interactions in Tuberous Sclerosis Complex Affect the Synaptic Balance in 2D and Organoid Cultures. Cells, 10(1), 134.

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Directed Differentiation of hAECs to Cortical Neurons

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We followed the protocol modified from Lippman et al. [12 (link)] that consists in proliferation and differentiation stages. At the first stage, P1 hAEC were incubated for 8 days in serum-free Insulin-Transferrin-Selenite medium (Life Technologies) supplemented with 1% antibiotic-antimycotic (basal medium), and six conditions were tested: 1) basal medium, 2) 10 ng/ml basic Fibroblast Growth Factor (bFGF; Peprotech), 3) bFGF plus 10 ng/ml of SB431542 (Stemgent, Cambridge, MA, USA), 4) bFGF plus 10 ng/ml Noggin (Sigma-Aldrich, St. Louis, MO, USA), 5) bFGF plus 10 ng/ml EGF and 6) bFGF, EGF, SB431542 and Noggin. At the differentiation stage, the cells were cultured for 6 days in serum-free N2 medium (Life Technologies) in which all the factors were withdrawn.
To evaluate differentiation to the neural linage, the expression of Nestin was detected by immunofluorescence at the end of the proliferation stage. We tested for the progenitor of cortical neuron markers Otx2, β-III-tubulin, TBR2 and NeuN at the end of the differentiation stage.

García-Castro I.L., García-López G., Ávila-González D., Flores-Herrera H., Molina-Hernández A., Portillo W., Ramón-Gallegos E, & Díaz N.F. (2015). Markers of Pluripotency in Human Amniotic Epithelial Cells and Their Differentiation to Progenitor of Cortical Neurons. PLoS ONE, 10(12), e0146082.

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Neuronal Differentiation from iPSCs

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For neuronal differentiation, 20–25 million iPS cells were plated on day 0 onto a 15 cm plate in N2 medium (Gibco) with N2 supplement (Thermo Fisher), supplemented with GlutaMAX (Thermofisher Fisher), MEM nonessential amino acids (NEAA) (Thermo Fisher), 10 μM ROCK inhibitor (Selleckchem), and 2 μg ml⁻¹ doxycycline (Sigma Aldrich). N2 medium was changed once a day for two more days. On day 3, cells were replated onto freshly prepared dishes coated with freshly prepared poly-L-ornithine (Sigma Aldrich). Pre-neuron cells were cultured in i³Neuron Culture Media: BrainPhys media (StemCell Technologies) supplemented with B27 Plus Supplement (ThermoFisher Scientific), 10 ng ml⁻¹ BDNF (PeproTech), 10 ng ml⁻¹ NT-3 (PeproTech), 1 μg ml⁻¹ mouse laminin (Sigma Aldrich), and 2 μg ml⁻¹ doxycycline (Sigma Aldrich). i³Neurons were then fed by half media and collected in different time points after neuronal induction.

Matos-Rodrigues G., van Wietmarschen N., Wu W., Tripathi V., Koussa N., Pavani R., Nathan W., Callen E., Belinky F., Mohammed A., Napierala M., Usdin K., Ansari A.Z., Mirkin S.M, & Nussenzweig A. (2022). S1-END-seq Reveals DNA Secondary Structures In Human Cells. Molecular cell, 82(19), 3538-3552.e5.

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Directed Differentiation of hPSCs to Neurons

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hPSCs were dissociated into single cells and seeded onto Matrigel (Corning, Corning, NY)-coated culture dishes. To initiate differentiation, the maintenance medium was replaced with knockout serum replacement medium in combination with N2 medium (Gibco). Medium was supplemented with 200 nM LDN193189 (STEMCELL Technologies, Vancouver, BC, Canada) and 10 μM SB431542 (Tocris, Ellisville, MO) and 2 μM each purmorphamine (Tocris) and RA (Sigma, St. Louis, MO). To support neuronal differentiation, the medium was switched at day 11 into NB/B27 medium (Gibco) supplemented with 20 ng/mL BDNF (Peprotech, London, UK). At day 20, the cells were passaged at a 1:2 ratio onto freshly prepared Matrigel-coated dishes.

Valiulahi P., Vidyawan V., Puspita L., Oh Y., Juwono V.B., Sittipo P., Friedlander G., Yahalomi D., Sohn J.W., Lee Y.K., Yoon J.K, & Shim J.W. (2021). Generation of caudal-type serotonin neurons and hindbrain-fate organoids from hPSCs. Stem Cell Reports, 16(8), 1938-1952.

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Sphere Formation Assay for Stem Cells

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Cells were seeded at 1 × 10³ cells/well in six-well ultralow attachment plates (Corning Inc., Corning, NY, USA) in DMEM/F12 (Invitrogen, Carlsbad, CA) supplemented with N2 medium (Invitrogen), human EGF (10 ng/ml, Peprotech, USA), and human bFGF (10 ng/ml, Peprotech). After culturing for 14 days, the total number of spheres was counted.

Zhang W., Yu F., Weng J., Zheng Y., Lin J., Qi T., Wei Y., Wang D, & Zeng H. (2021). SOX12 Promotes Stem Cell-Like Phenotypes and Osteosarcoma Tumor Growth by Upregulating JAGGED1. Stem Cells International, 2021, 9941733.

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Neurite Outgrowth in PC12 Cells as a Model

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Connections between nerve cells are altered in AD. Thus, compounds that can promote the regeneration of these connections might be of particular benefit, thereby promoting the recovery of higher neuronal function. As a model for this property, we used neurite outgrowth in rat PC12 cells, a well-studied model system of neuronal differentiation. In response to neurotrophic factors such as nerve growth factor (NGF), PC12 cells undergo a series of physiological changes culminating in a phenotype resembling that of sympathetic neurons [12] (link). In this assay, PC12 cells (originally obtained from Greene [13] (link)) grown in high-glucose DMEM supplemented with 10% FCS and 5% horse serum were plated in 35 mm tissue culture dishes. After 3 days of growth, the medium was replaced with serum-free N2 medium (Invitrogen) and the cells were treated with the extracts/compounds. After 24 h the cells were scored for the presence of neurites. PC12 cells produce neurites much more rapidly when treated in N2 medium than when treated in regular growth medium. For each treatment, 100 cells in each of three separate fields were counted. Cells were scored positive if one or more neurites longer than one cell body diameter in length were observed [14] (link).

Fischer W., Currais A., Liang Z., Pinto A, & Maher P. (2018). Old age-associated phenotypic screening for Alzheimer's disease drug candidates identifies sterubin as a potent neuroprotective compound from Yerba santa. Redox Biology, 21, 101089.

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Cancer Stem Cell Spheroid Formation

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143B cells were inoculated at a density of 1000 cells/well in six-well ultra-low attachment plates (Corning). And the cells were cultured in DMEM/F12 medium (Gibco) containing N2 medium (Invitrogen), human EGF (20ng/ml, PeproTech) and human bFGF (20ng/ml, PeproTech) for 14 days. Spheres were observed in size and the number of spheres formed was calculated.

Guo L., Yan T., Guo W., Niu J., Wang W., Ren T., Huang Y., Xu J, & Wang B. (2022). Molecular subtypes of osteosarcoma classified by cancer stem cell related genes define immunological cell infiltration and patient survival. Frontiers in Immunology, 13, 986785.

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Sarcosphere Formation Assay

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The assay for the formation of Sarcosphere was performed in accordance with a previously described protocol. Briefly, seeding of 1 × 10³ cells were done in 6-well plate of ultralow attachment in DMEM culture medium along with human bFGF and human EGF (20 ng/mL each), and N2 medium (Invitrogen). After every 3 days the culture media was changed. Using inverted phase contrast microscopy in three random fields, after a 14-day culture, sarcospheres were quantitated.
Following treatment with compounds for the formation of primary sarcospheres, treatment was not applied to assay the formation of secondary sarcospheres. Every 3 days, changing of culture media was done. After two full weeks of culturing, quantification of sarcospheres was done by inverted phase contrast microscopy (three fields chosen randomly).

Tian T., Guo T., Zhen W., Zou J, & Li F. (2020). BET degrader inhibits tumor progression and stem-like cell growth via Wnt/β-catenin signaling repression in glioma cells. Cell Death & Disease, 11(10), 900.

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Differentiating iPSCs from SCA3 Patients

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The culture and differentiation of iPSC from SCA3 patients were performed as previously described [24 (link)]. Picked neural rosettes were cultured in suspension for 2 days in DMEM/F12, 2 mM l-glutamine, 1.6 g l⁻¹ glucose, 0.1 mg ml⁻¹ penicillin/streptomycin, and N2 supplement (1:100; Invitrogen), then dissociated with trypsin, plated onto poly-l-ornithine/laminin-coated plates, and propagated in N2 medium supplemented with B27 (1 μl ml⁻¹, Invitrogen), 10 ng ml⁻¹ FGF2, and 10 ng ml⁻¹ EGF (both from R&D Systems). Neuronal differentiation was induced by removing the growth factors FGF2 and EGF from the media and culturing the cells in Neurobasal medium supplemented with B27 (1:50, Invitrogen) and DMEM/F12 supplemented with N2 (1:100) mixed at a 1:1 ratio; 300 ng ml⁻¹ cAMP was added to the differentiation media. After 6 weeks, differentiation cells were harvested in TRIzol.

Wiatr K., Piasecki P., Marczak Ł., Wojciechowski P., Kurkowiak M., Płoski R., Rydzanicz M., Handschuh L., Jungverdorben J., Brüstle O., Figlerowicz M, & Figiel M. (2019). Altered Levels of Proteins and Phosphoproteins, in the Absence of Early Causative Transcriptional Changes, Shape the Molecular Pathogenesis in the Brain of Young Presymptomatic Ki91 SCA3/MJD Mouse. Molecular Neurobiology, 56(12), 8168-8202.

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Dopaminergic-like Cell Line Electroporation

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The mouse SN4741 dopaminergic-like cell line was cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS; Invitrogen), 1% penicillin–streptomycin (Invitrogen), and 1% l-glutamine (Invitrogen)^{47 (link)}. For the purpose of electroporation, cells were trypsinized and suspended. Mouse ventral midbrain primary cells were obtained as previously described^{48 ,49 (link)}. Briefly, ventral midbrain tissue was dissected from E11.5 mouse embryos, dissociated with collagenase/dispase (30 min at 37 °C on a rocking platform), followed by mechanical trituration. For electroporation, cell suspension were aliquoted and electroporated with different plasmids according to manufacturer’s manual (Ingenio^® from Mirus Bio, Madison, Wisconsin). The following constructs were used: pCAG-IRES-Egfp, pCAG-Zeb2-Egfp, pCAG-Egfp-Sponge, and pMiR-200c-CAG-Egfp. After electroporation, SN4741 cells were plated in warm medium (10% FBS in DMEM, supplemented with 1% l-glutamine). Primary cells were plated at a density of 100,000 cells cm⁻² in N2 medium (Ham’s F-12 medium and minimal essential medium, supplemented with HEPES, N2 supplement, and l-glutamine, all from Invitrogen) in 48-well plate. Cells were subjected to fixation for immunocytochemistry or RNA extraction 48 h after electroporation.

Yang S., Toledo E.M., Rosmaninho P., Peng C., Uhlén P., Castro D.S, & Arenas E. (2018). A Zeb2-miR-200c loop controls midbrain dopaminergic neuron neurogenesis and migration. Communications Biology, 1, 75.

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