N2 and b27 supplements

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N2 and B27 supplements are laboratory reagents used in cell culture media to support the growth and differentiation of cells, particularly stem cells and neuronal cells. N2 supplement provides a defined mixture of nutrients and growth factors, while B27 supplement provides a more complex formulation of essential components. Both supplements are commonly used in research and development applications to maintain and expand various cell types in vitro.

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B27 supplement (3208 citations) N2 supplement (1487 citations) N2 and B27 (67 citations) B27 and N2 supplements (33 citations) N2/B27 supplements (10 citations)

75 protocols using n2 and b27 supplements

Photoinitiated Encapsulation and Differentiation

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24 hours after encapsulation, culture media was replaced with 500 μL of Fluorobrite medium (Thermo Fischer Scientific), supplemented with N2 and B27 supplements (Thermo Fischer Scientific), 15 mM reduced glutathione (Sigma) and 1 mM LAP. The cell laden hydrogels were allowed to equilibrate for 30 minutes at 37°C, after which the hydrogels were irradiated with 365 nm light at 5 mW cm⁻² for 0–10 seconds. After irradiation, Fluorobrite media was replaced with differentiation media, composed of Advanced DMEM-F12 (Invitrogen) with N2 and B27 supplements (Thermo Fischer Scientific), Glutamax (Gibco), HEPES, penicillin-streptomycin, and supplemented with epidermal growth factor (50 ng mL⁻¹, R&D Systems), Noggin (100 ng mL⁻¹, PeproTech), R-Spondin conditioned media (5% v/v), and n-acetylcysteine (1μM, SigmaAldrich).

Hushka E.A., Yavitt F.M., Brown T.E., Dempsey P.J, & Anseth K.S. (2020). Relaxation of Extracellular Matrix Forces Directs Crypt Formation and Architecture in Intestinal Organoids. Advanced healthcare materials, 9(8), e1901214.

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Dox-NIL iMN Generation and Transduction

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To generate Dox-NIL iMNs, the Dox-NIL construct was integrated into the AAVS1 safe harbor locus of the control and C9-ALS patient iPSC lines using CRISPR/Cas9 editing (gRNA sequence shown in Supplementary Table 4). The Conversion of NIL-iMNs was performed in 96-well plates (2 × 10³ cells/well), which was sequentially coated with matrigel (1 hour) and laminin (2–4 hours) at room temperature. iPSCs were cultured in mTeSr and induced with 1 μg/ml doxycycline on day 1 after plating. On day 3 after plating, the culture was transduced with CTRL shRNA1, CTRL shRNA2, TMX2 shRNA1 and TMX2 shRNA2 with 5 μg/ml polybrene in N3 media containing DMEM/F12 (Life Technologies), 2% FBS, 1% penicillin/streptomycin, N2 and B27 supplements (Life Technologies), and 10 ng/ml each of GDNF, BDNF, and CNTF (R&D). For the detection of the motor neurons, the culture was transduced with Hb9::RFP reporter 48 hours after shRNA transduction. On day 5, primary mouse cortical glial cells from P1 ICR pups (male and female) were added to the transduced cultures in N3 media.

Kramer N.J., Haney M.S., Morgens D.W., Jovičić A., Couthouis J., Li A., Ousey J., Ma R., Bieri G., Tsui C.K., Shi Y., Hertz N.T., Tessier-Lavigne M., Ichida J.K., Bassik M.C, & Gitler A.D. (2018). CRISPR-Cas9 screens in human cells and primary neurons identify modifiers of C9orf72 dipeptide repeat protein toxicity. Nature genetics, 50(4), 603-612.

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DUX Protein Expression and Regulation in mES Cells

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E14 wild-type mES cells were cultured on 0.1% gelatin-coated plates with mouse embryonic fibroblast feeder cells in LIF/2i medium (1:1 mix of DMEM/F12 (11320-033, Gibco) and Neurobasal medium (21103-049, Gibco) containing 1× N2 and B27 supplements (17502-048/17504-044, Life Technologies), 100 µM non-essential amino acids (GNM71450, GENOM), and 1000 U/ml LIF (PEPRO TECH), 1 μM PD03259010 and 3 μM CHIR99021 (STEMCELL Technologies) and 100 U/ml penicillin, 100 µg/ml streptomycin (15140-122, Gibco). For primed state media, 20 ng/ml Activin, 10 ng/ml FGF2, and 1% KSR were added to the 1:1 DMEM/F12 and Neurobasal medium containing N2 and B27. To investigate DUX binding, an N-terminal FLAG-DUX protein was expressed in our clonal cell lines. In control group, mES cells were treated with doxycycline for 12 h to induce FLAG-DUX expression and then treatment of negative-control Silencer Select siRNA. In siDux group, mES cells were treated with doxycycline for 12 h and then siDux for two days. In CX-5461 treatment group, mES cells were treated with doxycycline for 12 h and then treatment of CX5461. In siDux+CX-5461 group, mES cells were treated with doxycycline for 12 h and then siDux for 2 days followed by treatment of CX-5461.

Yu H., Sun Z., Tan T., Pan H., Zhao J., Zhang L., Chen J., Lei A., Zhu Y., Chen L., Xu Y., Liu Y., Chen M., Sheng J., Xu Z., Qian P., Li C., Gao S., Daley G.Q, & Zhang J. (2021). rRNA biogenesis regulates mouse 2C-like state by 3D structure reorganization of peri-nucleolar heterochromatin. Nature Communications, 12, 6365.

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Reprogramming Fibroblasts to Induced Motor Neurons

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Reprogramming was performed in 96-well plates (8 × 10³ cells/well) that were sequentially coated with gelatin (0.1%, 1 hour) and laminin (2–4 hours) at room temperature. To enable efficient expression of the transgenic reprogramming factors, iPSCs were cultured in fibroblast medium (DMEM + 10% FBS) for at least 48 hours and either used directly for retroviral transduction or passaged before transduction for each experiment. 7 iMN factors were added in 100–200 μl fibroblast medium per 96-well well with 5 μg/ml polybrene. For iMNs, cultures were transduced with lentivirus encoding the Hb9::RFP reporter 48 hours after transduction with transcription factor-encoding retroviruses. On day 5, primary mouse cortical glial cells from P1 ICR pups (male and female) were added to the transduced cultures in glia medium containing MEM (Life Technologies), 10% donor equine serum (HyClone), 20% glucose (Sigma-Aldrich), and 1% penicillin/streptomycin. On day 6, cultures were switched to N3 medium containing DMEM/F12 (Life Technologies), 2% FBS, 1% penicillin/streptomycin, N2 and B27 supplements (Life Technologies), 7.5 μM RepSox (Selleck), and 10 ng/ml each of GDNF, BDNF, and CNTF (R&D). The iMN neuron cultures were maintained in N3 medium, changed every other day, unless otherwise noted.

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Glioblastoma Cell Culture and Maintenance

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GBM1 and GBM4 (a gift from H. Wurdak) were maintained as described previously (Wurdak et al., 2010 (link)). They were cultured in Neural Basal medium consisting of Neurobasal Medium (Life Technologies), N2 and B27 supplements (Life Technologies, 0.5× each), recombinant basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) (Peprotech and R&D Systems, respectively, 40 ng/mL). GBM4UCL cells (a gift from S. Brandner, UCL Institute of Neurology) were cultured in DMEM/F12 (1:1 mixture), with 15 mM HEPES and 2 mM L-glutamine (Sigma), 1× B27, and 20 ng/mL each of recombinant EGF and bFGF. GSCs were cultured on laminin-coated (2 μg/mL; Sigma-Aldrich) surfaces. NHAs were purchased from Lonza and grown in AGM astrocyte growth medium (Lonza). U87-MG (ECACC) cells were grown in DMEM supplemented with 2 mM L-glutamine (Sigma) and 10% FBS (Biosera). BMP4 was obtained from Life Technologies. Protease inhibitor cocktail and trypsin were purchased from Sigma-Aldrich. The RAD51 inhibitors B02 and RI-1 were purchased from Sigma-Aldrich and Merck, respectively, and stored as 10 mM stock solutions in DMSO.

King H.O., Brend T., Payne H.L., Wright A., Ward T.A., Patel K., Egnuni T., Stead L.F., Patel A., Wurdak H, & Short S.C. (2017). RAD51 Is a Selective DNA Repair Target to Radiosensitize Glioma Stem Cells. Stem Cell Reports, 8(1), 125-139.

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Direct Reprogramming of Induced Neurons

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Reprogramming was performed in 96-well plates (8 × 10³ cells/well) or 13mm plastic coverslips (3.2 × 10⁴ cells/coverslip) that were sequentially coated with gelatin (0.1%, 1 hour) and laminin (2–4 hours) at room temperature. To enable efficient expression of the transgenic reprogramming factors, iPSCs were cultured in fibroblast medium (DMEM + 10% FBS) for at least 48 hours and either used directly for retroviral transduction or passaged before transduction for each experiment. 7 iMN factors or 5 iDA factors were added in 100–200 µl fibroblast medium per 96-well well with 5 μg/ml polybrene. For iMNs, cultures were transduced with lentivirus encoding the Hb9::RFP reporter 48 hours after transduction with transcription factor-encoding retroviruses. On day 5, primary mouse cortical glial cells from P1 ICR pups (male and female) were added to the transduced cultures in glia medium containing MEM (Life Technologies), 10% donor equine serum (HyClone), 20% glucose (Sigma-Aldrich), and 1% penicillin/streptomycin. On day 6, cultures were switched to N3 medium containing DMEM/F12 (Life Technologies), 2% FBS, 1% penicillin/streptomycin, N2 and B27 supplements (Life Technologies), 7.5 µM RepSox (Selleck), and 10 ng/ml each of GDNF, BDNF, and CNTF (R&D). The iMN and iDA neuron cultures were maintained in N3 medium, changed every other day, unless otherwise noted.

Shi Y., Lin S., Staats K.A., Li Y., Chang W.H., Hung S.T., Hendricks E., Linares G.R., Wang Y., Son E.Y., Wen X., Kisler K., Wilkinson B., Menendez L., Sugawara T., Woolwine P., Huang M., Cowan M.J., Ge B., Koutsodendris N., Sandor K.P., Komberg J., Vangoor V.R., Senthilkumar K., Hennes V., Seah C., Nelson A.R., Cheng T.Y., Lee S.J., August P.R., Chen J.A., Wisniewski N., Victor H.S., Belgard T.G., Zhang A., Coba M., Grunseich C., Ward M.E., van den Berg L.H., Pasterkamp R.J., Trotti D., Zlokovic B.V, & Ichida J.K. (2018). Haploinsufficiency Leads to Neurodegeneration in C9ORF72 ALS/FTD Human Induced Motor Neurons. Nature medicine, 24(3), 313-325.

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Dox-NIL iMN Generation and Transduction

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Glioblastoma Stem Cell Propagation and Notch Inhibition

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GBM tissue samples were obtained from patients undergoing surgical treatment at Nagoya University hospital, Japan, after they provided written informed consent. Total RNA of normal brain was purchased from BioChain (Hayward, CA, USA). The procedures used for derivation of GSCs (1228-GSC and 222-GSC) were as described previously5 (link). In brief, dissociated tumour cells were cultured in Neurobasal Medium (Life Technologies, Carlsbad, CA) containing N2 and B27 supplements (Life Technologies), along with human recombinant basic fibroblast growth factor and epidermal growth factor (20 ng/ml each; R&D Systems, Minneapolis, MN, USA). Serum-differentiated GSCs were established by culturing GSCs in DMEM (Life Technologies) containing 10% fetal bovine serum5 (link). Serially transplanted GSCs were generated according to previously published methods1 (link). GSCs were routinely tested for mycoplasma contamination and used in the experiments. For blocking of Notch signalling in GSCs, the γ-secretase inhibitor DAPT (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenyl glycine t-butyl ester), Sigma-Aldrich, St Louis, MO, USA) or RO4929097 (Selleck Chemicals, Houston, TX, USA) were added, and the medium was changed every other day. For in vivo studies, RO4929097 was formulated as a suspension in 1% carboxymethyl cellulose with 0.2% Tween 80 for oral administration.

Katsushima K., Natsume A., Ohka F., Shinjo K., Hatanaka A., Ichimura N., Sato S., Takahashi S., Kimura H., Totoki Y., Shibata T., Naito M., Kim H.J., Miyata K., Kataoka K, & Kondo Y. (2016). Targeting the Notch-regulated non-coding RNA TUG1 for glioma treatment. Nature Communications, 7, 13616.

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Embryonic Cortical Neural Precursor Isolation

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Primary culture of embryonic cortical NPCs was performed as described (Li et al., 2017 (link)). In brief, E11.5 or E12.5 mouse cortices (dorsal forebrain) tissues were washed with and minced in filter-sterilized hibernation buffer (30 mmol/L KCl; 5 mmol/L NaOH; 5 mmol/L NaH₂PO₄; 5.5 mmol/L glucose; 0.5 mmol/L MgCl₂; 20 mmol/L Na-pyruvate; 200 mmol/L Sorbitol, pH 7.4) followed by dissociating into single cells using pre-warmed Papain (Worthington Biochemical) enzyme solution (1× DMEM; 1 mmol/L Na-pyruvate; 1 mmol/L L-glutamine; 1 mmol/L N-acetyl-L-cysteine; 20 U/mL Papain; 12 μg/mL DNase I). Dissociated cells were cultured using serum-free media consisting of DMEM/F12 media (Life Technologies), N2 and B27 supplements (1×, Life Technologies), 1 mmol/L Na-pyruvate, 1 mmol/L N-acetyl-L-cysteine (NAC), human recombinant FGF2 and EGF (20 ng/mL each; Life Technologies). For adherent cortical cultures in Figs. 2C, S3A and S3B, cells were maintained on poly-L-lysine coated plates with the presence of 20 ng/mL FGF2 for 24 h followed by differentiation (FGF2 withdrawal) for 48 h. For sphere culture in Fig. 3F, cells were cultured with the presence of EGF and FGF2 for 1 week. For clonal culture in Fig. 6A–G, cells were maintained 72 h with the presence of 20 ng/mL FGF2 for 72 h (4 × 10⁴ cells per well in 24-well plates).

Li W., Shen W., Zhang B., Tian K., Li Y., Mu L., Luo Z., Zhong X., Wu X., Liu Y, & Zhou Y. (2019). Long non-coding RNA LncKdm2b regulates cortical neuronal differentiation by cis-activating Kdm2b. Protein & Cell, 11(3), 161-186.

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Retinal Photoreceptor Differentiation from iPSCs

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Differentiation into retinal photoreceptors was performed as previously described [26 (link)], using iPSCs cultured in feeder-free conditions on Matrigel in mTESR^TM medium. The cells were dissociated enzymatically and plated onto Matrigel-covered dishes in neural differentiation medium containing N2 and B27 supplements (Life Technologies). After settling for an hour, adhered cells were covered in a 2% Matrigel solution. The following day, and thereafter every second day, the medium was replaced with neural differentiation medium without Matrigel. From day 10 the medium was supplemented with 3nM recombinant SHH (R&D Systems), 50ng/μl acidic fibroblast growth factor (αFGF) (R&D Systems), 10ng/μl basic fibroblast growth factor (bFGF) (Miltenyi), 1mM taurine and 500nM retinoic acid (both Sigma Aldrich). After 30 days of culture characterisation was performed by immunocytochemistry and qRT-PCR (for antibodies and primers, see S1 and S2 Tables). Each patient and control line underwent two rounds of differentiation, with each time point analysed in biological duplicate.

Burman R.J., Watson L.M., Smith D.C., Raimondo J.V., Ballo R., Scholefield J., Cowley S.A., Wood M.J., Kidson S.H, & Greenberg L.J. (2021). Molecular and electrophysiological features of spinocerebellar ataxia type seven in induced pluripotent stem cells. PLoS ONE, 16(2), e0247434.

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