N 2 supplement 100x

Manufactured by Thermo Fisher Scientific

Sourced in United States

The N-2 Supplement 100X is a concentrated solution of insulin, transferrin, selenium, and other components that supports the growth and maintenance of neuronal and other cell types in vitro. It is designed to be added to cell culture media to supplement the nutritional requirements of cells.

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13 protocols using n 2 supplement 100x

Neuronal Differentiation from NPCs

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Neuronal Precursor Cells (NPC) were maintained in NPC media, containing DMEM/F12 with Glutamax (Cat. #10565010, ThermoFisher Scientific, MA), 1X B27 Supplement, minus Vitamin A (Cat. #12587070, ThermoFisher Scientific, MA), 1X N-2 Supplement (100X) (Cat. #17502048, ThermoFisher Scientific, MA), Laminin (Cat. #A25607, ThermoFisher Scientific, MA) and FGF2 (Joint Protein Central, Korea).
Differentiation of NPCs to neurons was performed for varying differentiation times including 1, 2, 3, and 4 weeks with media change every 48hrs. Differentiation media contained DMEM/F12 with Glutamax (Cat. #10565010, ThermoFisher Scientific, MA), 1X B27 Supplement (Cat. #17502048, ThermoFisher Scientific, MA), 1X N-2 Supplement (100X) (Cat. #17502048, ThermoFisher Scientific, MA), Laminin (Cat. #A25607, ThermoFisher Scientific, MA), BDNF (Cat. #450–02-1mg, Peprotech, NJ, United States), GDNF (Cat. #450–10-1mg, Peprotech, NJ, United States), 1 mM dibutryl cyclicAMP (cAMP) (Cat. #1141, Tocris Bioscience, UK (and 200 nM ascorbic acid (Cat. #72132, StemCell Technologies, United States).

Mishra P., Sivakumar A., Johnson A., Pernaci C., Warden A.S., El-Hachem L.R., Hansen E., Badell-Grau R.A., Khare V., Ramirez G., Gillette S., Solis A.B., Guo P., Coufal N, & Cherqui S. (2024). Gene editing improves endoplasmic reticulum-mitochondrial contacts and unfolded protein response in Friedreich’s ataxia iPSC-derived neurons. Frontiers in Pharmacology, 15, 1323491.

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Human Astrocyte Culture Protocol

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Human astrocytes (SciencCell research laboratories San Diego CA Catalog #1800) were seeded at a density of 10⁵ in 6 well plates (BD Bioscience) in growth media which contained 89% Dulbecco’s Modified Eagle Medium, 1% N-2 Supplement 100X, 10% Fetal Bovine serum (all Life Technologies). Once cells had reached 70% confluence they were lysed for RNA and protein isolation. Growth media was removed from wells before 200 ul of Tripure (Roche) was added per well of a 6 well plate and incubated at room temperature for 5 minutes with occasional gentle agitation. For protein isolation 150 ul of protein lysis buffer was added per well and incubated on ice for 5 minutes with occasional gentle agitation.

Durcan P.J., Conradie J.D., Van deVyver M, & Myburgh K.H. (2014). Identification of novel Kirrel3 gene splice variants in adult human skeletal muscle. BMC Physiology, 14, 11.

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Isolation of Murine Dorsal Root Ganglia Neurons

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Neuron cultures were obtained from C57BL/6 male mice of 8 weeks. All experimental procedures were carried out in compliance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines and in accordance with National Institute of Health guidelines for animal care and use of Laboratory animals (DL 2016, Italian Ministry of Health approval protocol 919/2015-PR). A complete dorsal root ganglia (DRG) pool was collected in F12 medium (Euroclone, Italy) from each mouse. DRG were digested for 1 h with 12.5 mg/ml collagenase (Sigma Aldrich, USA) and 10 mg/ml DNase (Sigma Aldrich, USA) and then mechanically triturated. A BSA gradient (30% BSA, 70% F12 medium) was used to isolate neurons that were then resuspended in BS medium (F12 medium supplemented with 1% N2 supplement 100X (Life Technologies, UK), 1% BSA (Sigma Aldrich, USA), 1% Penicillin and Streptomycin 100X (Euroclone, Italy), 1% L-glutamine 100X (Euroclone, Italy) and seeded in a single drop on poly-l-lysine (Sigma Aldrich, USA) coated dishes. After 24 h, neurons cultures were treated with 10⁻⁵ M 2′-Deoxy-5-fluorouridine (FuDR) (Sigma, USA) to remove satellite cells.

Malacrida A., Semperboni S., Di Domizio A., Palmioli A., Broggi L., Airoldi C., Meregalli C., Cavaletti G, & Nicolini G. (2021). Tubulin binding potentially clears up Bortezomib and Carfilzomib differential neurotoxic effect. Scientific Reports, 11, 10523.

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Colorectal Tumor Organoid Culture

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Colorectal tumor tissues were obtained from The Sixth Affiliated Hospital of Sun Yat‐sen University with informed consent and the study was approved by the ethical committee (SYSU‐IACUC‐2020‐000570). Fresh tumor tissues were dissociated into single‐cell suspensions with the Tumor Dissociation Kit (Miltenyi Biotec, 130‐095‐929). CRC organoids were cultured in CRC organoid medium.^[⁵⁷^] The composition of CRC organoid medium was: 14 mL advanced DMEM/F12 medium (Life Technologies) with 1% GlutaMax I 100x (Life Technologies), 1% Hepes (Life Technologies), 1% Penicillin Streptomycin 100x (Thermofisher), 0.1% Primocin (Invivogen), 2% B27 supplement 50x (Life Technologies), 3.5 µg R‐Spondin 3 (R&D), 2.8 µg EGF (Peprotech), 2.95 µg A83‐01 (Tocris), 1.4 µg Wnt3A (R&D), 46.4 µg SB202190 (Sigma), 2.24 mg N‐acetylcysteine (Sigma), 0.168 mg nicotinamide (Sigma), and 1% N2 supplement 100x (Life Technologies).

Yu Z., Deng P., Chen Y., Liu S., Chen J., Yang Z., Chen J., Fan X., Wang P., Cai Z., Wang Y., Hu P., Lin D., Xiao R., Zou Y., Huang Y., Yu Q., Lan P., Tan J, & Wu X. (2021). Inhibition of the PLK1‐Coupled Cell Cycle Machinery Overcomes Resistance to Oxaliplatin in Colorectal Cancer. Advanced Science, 8(23), 2100759.

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Differentiation of Embryonic Stem Cells into Neurons

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Three different lines of embryonic stem cells derived from wild type (wt) backgrounds R1, E14Tg2a and J1 were cultured and differentiated into neurons as described (Bibel et al., 2007 (link)). Briefly, after 4 days of embryoid body formation they are treated with 5 µM all-trans-retinoic acid (Sigma-Aldrich, Buchs, Switzerland) for additional 4 days. Embryoid bodies were dissociated and neuronal precursors were plated on poly-L-ornithine (Sigma)/laminin (Roche Inc., Buchs, Switzerland)—coated glass cover slips (Assistent, Karl Hecht GmbH, Sondheim/Rhön, Germany). At day in vitro (DIV) 0 and 1 neuronal precursors were cultured in neural medium containing DMEM/F12, N-2 Supplement (100X) and penicillin/streptomycin and 1 mM glutamine (all Invitrogen Inc., Lucerne, Switzerland). From DIV 2 the medium was changed to the differentiation medium containing Neurobasal® medium, B-27® Supplement (50X), N-2 Supplement (100X), 0.6 mM glutamine and penicillin/streptomycin (all Invitrogen).

Barth L., Sütterlin R., Nenniger M, & Vogt K.E. (2014). Functional differentiation of stem cell-derived neurons from different murine backgrounds. Frontiers in Cellular Neuroscience, 8, 49.

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Enhancing Gastric Epithelial Stemness

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GES-1 cells were cultured with RPMI-1640 medium containing 10% FBS (control), CM from hucMSCs or CM from macrophage-hucMSCs for 48 h at 37°C. Subsequently, GES-1 cells from all groups were harvested and plated at density of 2,000 cells/well into 24-well culture plates coated with 10% poly-HEMA (Sigma-Aldrich; Merck KGaA) solution in 100% ethanol and dried overnight at 56°C with a serum-free RPMI-1640 Glutamax medium (Invitrogen; Thermo Fisher Scientific, Inc.) containing 15 ng/ml EGF (Bioworld Technology, Inc.), 10 ng/ml FGF (Bioworld Technology, Inc.), 1:100 N-2 supplement 100X (Invitrogen; Thermo Fisher Scientific, Inc.), 0.3% glucose, 5 mg/ml gentamicin (Sigma-Aldrich; Merck KGaA), 50 IU/ml penicillin and 2.5 mg/ml amphotericin B (Sigma-Aldrich; Merck KGaA), respectively, all groups were incubated in humidified incubator at 37°C with 5% CO₂ for 15 days. Every 3 days, 50% of the spent medium was removed and replaced. The total number of spherical colonies (colonies with diameters large than 50 µm were counted) obtained was quantitated under an inverted microscope. This procedure was repeated at least three times.

Zhang Q., Chai S., Wang W., Wan C., Zhang F., Li Y, & Wang F. (2018). Macrophages activate mesenchymal stem cells to acquire cancer-associated fibroblast-like features resulting in gastric epithelial cell lesions and malignant transformation in vitro. Oncology Letters, 17(1), 747-756.

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Neuronal Cell Culture Protocol

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The following reagents were purchased from Thermo Fisher Scientific (Waltham, MA, USA): B-27^® supplement 50x, Fluo-4 AM cell permeant, formaldehyde 37%, GlutaMAX™ supplement, human recombinant epidermal growth factor (EGF), MEM non-essential amino acids solution 100x, N-2 supplement 100x, and phosphate buffered saline 1x (PBS). The following reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA): 2,5-hexanedione, γ-Aminobutyric acid (GABA), bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), Dulbecco’s Modified Eagle Medium (DMEM), endosulfan, methylmercury, penicillin-streptomycin solution 100x, Triton™ X-100, and TWEEN^® 20. Matrigel^® was purchased from Corning (Corning, NY, USA). Fetal calf serum (FCS) was purchased from ATCC (Manassas, VA, USA). Human recombinant fibroblast growth factor basic (bFGF) was purchased from Peprotech (Rocky Hill, NJ, USA). Tetrodotoxin (TTX) was purchased from Tocris Bioscience (Bristol, UK).

Wevers N.R., van Vught R., Wilschut K.J., Nicolas A., Chiang C., Lanz H.L., Trietsch S.J., Joore J, & Vulto P. (2016). High-throughput compound evaluation on 3D networks of neurons and glia in a microfluidic platform. Scientific Reports, 6, 38856.

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Optimizing Neural Stem Cell Culture

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Unless otherwise indicated, chemicals were purchased from Sigma-Aldrich (UK). Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 GlutaMAX
^TM (DMEM/F12, Thermo Fisher Scientific, 31331028), Neurobasal®-A Medium (Life Technologies, 10888022), foetal bovine serum (FBS, Life Technologies, 10270106), N-2 supplement 100x (Thermo Fisher Scientific, 17502048) and B-27 supplement 50x (Thermo Fisher Scientific, 12587010), human FGF-2 (Peprotech, AF-100-18B), EGF (PeproTech, 100-15) and PDGF-aa (Peprotech, 100-13A-10), and PDGF-aa (Peprotech, 100-13A-10), propidium iodide (PI) from Invitrogen and Allophycocyanin (APC) Annexin V from BD Pharmingen 550474 BD).

Helenes González C., Jayasinghe S.N, & Ferretti P. (2020). Bio-electrosprayed human neural stem cells are viable and maintain their differentiation potential. F1000Research, 9, 267.

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Cochlear Explant Culture Preparation

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The cochlea was dissected using sterile conditions under Nikon SM2 745 T Stereomicroscope (Nikon). Explant cultures were prepared from the cochleae of ZO1-EGFP transgenic mice. The cochleae were removed and placed in ice-cold PBS. Using two forceps the organ of Corti was gently freed from the capsule and separated from the stria vascularis. The tissue was oriented so that the apical surfaces of the hair cells were pointing down, directed toward the Matrigel Phenol Red Free solution (In vitro technologies, cat: FAL356237). Excess medium was removed and explanted tissue was allowed to attach to the Matrigel for 5–10 min in a 37 °C incubator with 5% CO₂ while avoiding drying of the tissue. After tissue attachment, Dulbecco’s modified Eagle’s medium (Biological industries, cat: 01-053-1 A) supplemented with N-2 Supplement (100X) (Thermo Fisher Scientific, cat: 17502001) and 1% FBS (Biological industries, cat: 04-007-1 A) was added gently. The plate was then placed in the 37 °C incubator of the microscope and as a control in the lab incubator. Cultures were kept for up to 48 h.

Cohen R., Amir-Zilberstein L., Hersch M., Woland S., Loza O., Taiber S., Matsuzaki F., Bergmann S., Avraham K.B, & Sprinzak D. (2020). Mechanical forces drive ordered patterning of hair cells in the mammalian inner ear. Nature Communications, 11, 5137.

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Neuroectodermal Differentiation Protocol

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Differentiation towards the neuroectodermal lineage was performed as previously described (Aguilo et al., 2017) . Briefly, 5x10 4 cells/ml were plated in low-attachment dishes in the presence of complete medium without LIF. Following EB formation for 2 days, EBs were cultured in media containing DMEM/F-12, 0.1 mM 2-mercaptoethanol, N2 supplement (100X) (ThermoFisher Scientific), B27 supplement (50X) (ThermoFisher Scientific), and penicillin/streptomycin (ThermoFisher Scientific) supplemented with 1 μM RA (Sigma-Aldrich) for 4 days to allow neuroectodermal differentiation. On day 6, EBs were transferred onto tissue culture dishes pre-coated with 0.1% gelatin and neuroectodermal differentiation media was replenished every 48 hours. Differentiated cells were harvested for total RNA, nuclear protein extraction and immunostaining analysis at the indicated time points. Cell cultures were maintained at 37°C with 5% CO2 in a humidified incubator.

Malla S., Bhattarai D.P., Melguizo‐Sanchis D., Atanasoai I., Groza P., Román Á., Zhu D., Lee D., Kutter C., & Aguiló F. (2021). ZFP207 controls pluripotency by multiple post-transcriptional mechanisms.

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