N2 supplement

Manufactured by R&D Systems

Sourced in United States

The N2 Supplement is a cell culture media supplement that provides a defined, serum-free formulation of growth factors and other components to support the growth and expansion of neural stem/progenitor cells.

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N2-max supplement (4 citations) N2-plus supplement (3 citations)

18 protocols using n2 supplement

Generating Mouse Intestinal Enteroids

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Small intestinal enteroids were generated from 4 to 8 week old wild type C57BL6 mice as previously described [51 (link)]. Enteroids were cultured in Advanced DMEM/F12 (Life Technologies 12,634) supplemented with 1x N-2 supplement (R&D Systems AR009), 1x B27 supplement (Gibco 17,504), 10 mM HEPES (Thermo Fisher Scientific15630080), 1x Glutamax (Gibco 15,630,080), 100 U/mL Penicillin-Streptomycin (Gibco 10,378,016), 50 ng/mL recombinant mouse EGF (Peprotech 315–09), 50 ng/mL recombinant murine noggin (Peprotech 250–38), and 50 ng/mL recombinant mouse r-spondin CHO-expressed (R&D Systems 7150-RS). Organoid cultures were passaged a minimum of one time prior to experimentation.

Dougherty M.W., Kudin O., Mühlbauer M., Neu J., Gharaibeh R.Z, & Jobin C. (2020). Gut microbiota maturation during early human life induces enterocyte proliferation via microbial metabolites. BMC Microbiology, 20, 205.

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Organoid Culture from Intestinal Crypts

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For construction of organoids, 200–500 crypts per well were suspended in Matrigel (Corning) as described.^{58 (link)} Complete ENR medium (all components from Thermo Fisher Scientific unless noted) were comprised of advanced DMEM/F12 (Gibco), antibiotic-antimycotic (×100), 1 mM N-acetyl cysteine (Sigma-Aldrich), B27 supplement, N2 supplement, EGF, Noggin (R&D Systems), R-spondin-1-conditioned medium, and Y-27632 (Sigma). Y-27632 was added to the ENR medium for the first 48–72 h of culture only and then removed during the medium change. The ENR medium was replaced every 2 to 3 days. Colon organoids were cultured in the ENR medium supplemented with Wnt3 conditioned media (WENR). Human colon organoids were cultured in WENR medium supplemented with gastrin, nicotinamide, A83-01, and SB202190 (all from Sigma) as described.^{59 (link)} Isolated ISCs and Paneth cells were co-cultured in ENR medium supplemented with Jagged-1 (1 µM; Anaspec). Wnt-C59 (50 µM; Abcam) was used as a porcupine (PORCN) inhibitor. The surface areas of SI and colon organoids were measured microscopically by taking several random non-overlapping photos of organoids in a well using an inverted microscope (Carl Zeiss). Each photo was analyzed using ImageJ software (NIH) and the Zen image program (Carl Zeiss). Organoid perimeters for area measurements were defined manually using automated ImageJ software.

Kim S., Shin Y.C., Kim T.Y., Kim Y., Lee Y.S., Lee S.H., Kim M.N., O E., Kim K.S, & Kweon M.N. (2021). Mucin degrader Akkermansia muciniphila accelerates intestinal stem cell-mediated epithelial development. Gut Microbes, 13(1), 1892441.

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Expansion of Mouse Embryonic Stem Cells

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Feeder dependent female WT and Ptbp1^−/− BL6 mouse ES cells were expanded on inactive male murine embryonic fibroblast (MEF) feeders on 0.1% gelatinized tissue culture plates in DMEM media (Gibco cat. no. 10313039) supplemented with 15% ESC grade FBS (Gibco cat. no. 10439024), 1% nucleosides (EMD Millipore cat. no. ES008D), 1% Glutamax (Gibco cat. no. 35050061), 0.1 mM β-mercaptoethanol (Acros Organics cat. no. 125472500), and 1000 U/ml mLIF (EMD Millipore cat. no. ESG1106). Feeder independent female WT and Ptbp1^−/− BL6 mouse ES cells, and F1 2-1 mouse ES cells were expanded on 0.1% gelatinized tissue culture plates in 2i culture media containing 50% Neurobasal (Gibco cat. no. 21103049) and 50% DMEM/F12 (Gibco cat. no. 11320082) supplemented with 1% B27 + RA (Gibco cat. no. 17504044), 1% N2 Supplement (R&D System cat.no. AR009), 1% Glutamax, 7.4 mM B27 Fraction V (Gibco cat. no. 15260037), 1% Penicillin-Streptomycin (GE Healthcare Life Sciences cat. no. SV30010), 3 μM CHIR99021 (Sigma cat. no. SML1046), 1 μM PD0325901 (Selleckchem cat. no. S1036), 150 μM 1-thioglycerol (Sigma cat. no. M6145) and 1000 U/ml mLIF (Gemini Bio-Products cat. no. 400–495). To assist initial attachment, 2i medium was supplemented with 2% ESC grade FBS for the first 24 h before switching to serum free.

Stork C., Li Z., Lin L, & Zheng S. (2018). Developmental Xist induction is mediated by enhanced splicing. Nucleic Acids Research, 47(3), 1532-1543.

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Differentiation of Adult Neural Cells

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Adult NC cells were cultured on poly-L ornithine (100 ng/ml; Sigma)/laminin (10 mg/ml; EMD Millipore, Billerica, MA) coated dishes and exposed to neuron differentiation media containing Neurobasal plus medium (Thermo Fisher Scientific) with BMP2 (10 ng/ml; R&D systems, Minneapolis, MN), SB431542 (10 μM), B27 plus (Thermo Fisher Scientific), N2 supplement (R&D systems), Brain-derived neurotrophic factor (BDNF; 10 ng/ml; Thermo Fisher Scientific), Glial cell-derived neurotrophic factor (GDNF;10 ng/ml; Sigma), Nerve growth factor (NGF; 10 ng/ml; R & D systems), Neurotrophin 3 (NT3; 10 ng/ml; Sigma), ascorbic acid (200 μM; Sigma) and cyclic adenosine monophosphate (0.5 mM cAMP; Sigma), CHIR 99021 (0.5 μM, only on day 1; Sigma), 2% FBS (from day 1–5), IWP-4 (100 nM days 4–6; 1 μM thereafter; Tocris Bioscience, Minneapolis, MN).

Moghadasi Boroujeni S., Koontz A., Tseropoulos G., Kerosuo L., Mehrotra P., Bajpai V.K., Selvam S.R., Lei P., Bronner M.E, & Andreadis S.T. (2019). Neural crest stem cells from human epidermis of aged donors maintain their multipotency in vitro and in vivo. Scientific Reports, 9, 9750.

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Derivation and Expansion of Neuropotent NPCs

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Derivation of neuropotent self-renewing NPCs was performed as previously described [30 (link)]. Mouse iPSCs were harvested, resuspended in neural induction medium (DMEM/F12 and Neurobasal medium) (1:1, Invitrogen), 0.5× N2 supplement (Invitrogen), 1× B27 supplement (Invitrogen), 1× antibiotic, and antimitotic stock solution (Invitrogen), and transferred to 0.1% gelatin-coated plates. Culture medium was replaced every day. After 12 days, cells were harvested by Accutase solution (Millipore, Burlington, MA, USA), resuspended in neural progenitor expansion media (NPEM) (DMEM/F12, 1% N2 supplement, 20 ng/mL EGF and 20 ng/mL bFGF) (R&D Systems) and 1x antibiotic and antimitotic stock solution and transferred to Geltrex LDEV-free reduced growth factor basement membrane matrix- treated (1:100, Invitrogen) dishes. Medium was changed every other day.

Armijo E., Edwards G., Flores A., Vera J., Shahnawaz M., Moda F., Gonzalez C., Sanhueza M, & Soto C. (2021). Induced Pluripotent Stem Cell-Derived Neural Precursors Improve Memory, Synaptic and Pathological Abnormalities in a Mouse Model of Alzheimer’s Disease. Cells, 10(7), 1802.

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UHPLC Analysis of Pharmaceutical Compounds

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For ultra-high performance liquid chromatography (UHPLC) analysis, analytical-grade formic acid and UHPLC grade solvents were obtained from Fisher Scientific Ltd. (Loughborough, UK). FSA as an authentic standard chemical (STD) was purchased from ChemFaces (Wuhan, Hubei, China), and its chemical purity was > 98% according to the manufacturer’s information sheet. Oxaliplatin, bortezomib and amifostine were purchased from TOCRIS Bioscience (Bristol, UK), Cell Signaling Technology (Danvers, MA, USA) and Santa Cruz Biotechnology (Dallas, TX, USA), respectively. Cisplatin, paclitaxel, docetaxel, and vincristine were purchased from Sigma-Aldrich Co (St. Louis, MO, USA). Recombinant rat nerve growth factor (NGF) and N2 supplement were purchased from R&D systems (Minneapolis, MN, USA) and Thermo Fisher Scientific (Waltham, MA, USA), respectively. A rabbit antibody against PGP9.5 and a goat anti-rabbit IgG Alexa Flour 488-labeled antibody were purchased from Millipore (Temecula, CA, USA) and Abcam (Brandford, CT, USA), respectively.

Yi J.M., Shin S., Kim N.S, & Bang O.S. (2019). Ameliorative effects of aqueous extract of Forsythiae suspensa fruits on oxaliplatin-induced neurotoxicity in vitro and in vivo. BMC Complementary and Alternative Medicine, 19, 339.

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Somitoid Induction from Stem Cells

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The N2B27 medium is a mixture of DMEM/F12 (Gibco, 21331020) with 1x N2 supplement (R&D, AR009) and Neurobasal medium (Gibco, 21103049) with 1x B27 supplement (Gibco, 17504-044) at a 1:1 ratio. The N2B27 medium was also supplemented with 2 mM Glutamax (Gibco, 35050-038), 0.1 mM nonessential amino acids (Gibco, 11140-035), 1 mM sodium pyruvate (Gibco, 11360-039), and penicillin/streptomycin (Gibco, 15140-122).
The Somitoid induction medium is the N2B27 medium containing 10 μM SB431542 (Sigma, S4317), 8-10 μM CHIR99021 (Sigma, SML1046), 2 μM DMH1 (Sigma, D8946), and 20 ng/ml bFGF (PeproTech, AF-100-18B).

Sanaki-Matsumiya M., Matsuda M., Gritti N., Nakaki F., Sharpe J., Trivedi V, & Ebisuya M. (2022). Periodic formation of epithelial somites from human pluripotent stem cells. Nature Communications, 13, 2325.

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Directed Differentiation of Definitive Endoderm

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Stem cells were handled as stated above, but only
0.5×10⁵ cells were plated. Differentiation was
initiated when cells reached ~40% confluency, approximately 48 hours
after plating. Cells were treated with 100ng/ml Activin A for 3 consecutive
days in RPMI 1640 (Invitrogen) with increasing concentrations of
tetracycline-free FBS (0%, 0.2%, 2% on day 1, 2, 3, respectively).
Definitive endoderm was then incubated for four days in DMEM-F12 containing
2% tetracycline-free FBS, 400ng ml⁻¹ FGF4, and 3μM
CHIR99021 (Stemgent). Spheroids were then collected and embedded in a
50μl bubble of Matrigel (BD Bioscience). The bubble was allowed to
solidify for 30 min at 37C, and then overlaid with gut media (advanced
DMEM/F12 (Invitrogen), L-glutamine, 10μM HEPES, 1× N2
supplement (R&D Systems), 1× B27 (Invitrogen), pen/strep, and
100ng ml⁻¹ EGF (R&D Systems)). Gut media was replaced
every four days.

Zhang X., McGrath P.S., Salomone J., Rahal M., McCauley H.A., Schweitzer J., Kovall R., Gebelein B, & Wells J. (2019). A comprehensive structure-function study of Neurogenin3 disease causing alleles during human pancreas and intestinal organoid development.. Developmental cell, 50(3), 367-380.e7.

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Cell Culture of Glioblastoma Models

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U87 and U1242 cells were grown in DMEM/10% FBS (GE Healthcare, Chicago, IL) with 1% penicillin/streptomycin (Thermo Fisher). GB30 neurosphere cells <20 passages were cultured in DMEM/F12 (Corning) with 1% N2 supplement, 1% penicillin/streptomycin, and 20 ng/mL bFGF and EGF (R&D Systems). All cells were incubated in 5% CO₂/air at 37°C. Patient-derived GB30 cells were obtained as previously described [7 (link)]. U87 cells were purchased from the American Type Culture Collection. STR profiling of GB30 and U1242 cells was performed at the University of Arizona Genetics Core for authentication.

Zumbar C.T., Usubalieva A., King P.D., Li X., Mifsud C.S., Dalton H.M., Sak M., Urio S., Bryant W.M., McElroy J.P., Farmer G, & Lehman N.L. (2018). The CNS penetrating taxane TPI 287 and the AURKA inhibitor alisertib induce synergistic apoptosis in glioblastoma cells. Journal of neuro-oncology, 137(3), 481-492.

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Culturing Adult Hippocampal Neural Stem Cells

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Adult hippocampal NSC cultures were derived as previously described (Blomfield et al., 2019 (link)). Briefly, 7-to-8-week-old mice were sacrificed and the DG dissected (previously described by Walker and Kempermann, 2014 (link)). For each new line, the two DGs of a single mouse of the desired genotype were dissociated using the Neural Tissue dissociation kit (P) (Milteny Biotec, 130-092-628). NSCs were amplified in nonadherent conditions as neurospheres in basal media [DMEM:F12+L-glutamine+sodium bicarbonate (Gibco, 11320)]+1 mg/ml KCl (Sigma, P5405)+2 mg/ml bovine serum albumin (BSA; Sigma, A9056)+1× Neurocult Supplement (Stem Cell Technologies, 05701)+1× penicillin/streptomycin (Thermo Fisher Scientific, 15140)+10 ng/ml FGF2 (Protech, 450-33)+20 ng/ml EGF (Protech, 315-09)+2 µg/ml heparin (Sigma, H3393) for at least two passages before dissociation to adherent cultures. NSCs were propagated in basal media [DMEM/F-12+Glutamax (Invitrogen 31331-093)]+1× N2 Supplement (R&D Systems, AR009)+1× penicillin/streptomycin+2 µg/ml laminin (Sigma, L2020)+5 µg/ml heparin+20 ng/ml FGF2. Neurospheres and adherent NSCs were incubated at 37°C, 5% CO₂ and routinely tested for mycoplasma contamination.

Austin S.H., Gabarró-Solanas R., Rigo P., Paun O., Harris L., Guillemot F, & Urbán N. (2021). Wnt/β-catenin signalling is dispensable for adult neural stem cell homeostasis and activation. Development (Cambridge, England), 148(20), dev199629.

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