N2 neural supplement

Manufactured by Thermo Fisher Scientific

Sourced in United States

N2 neural supplement is a lab product designed to support cell culture media for neural cells. It provides a defined combination of nutrients, hormones, and other components essential for the growth and maintenance of neural cells in vitro.

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8 protocols using n2 neural supplement

Neural Retina Cell Isolation and Culture

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RPCs were obtained from the fresh neural retina that isolated from postnatal day 1 C57BL/6 mice^{38 (link)}, and cells from the fifth passage or sixth passage were used in this study. The proliferation medium contains 20 ng/ml recombinant epidermal growth factor (EGF, Invitrogen, Carlsbad, CA, USA), advanced Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Invitrogen), 2 mM l-glutamine (Invitrogen) and 1% N2 neural supplement (Invitrogen)^{6 (link)}. The differentiation medium consists of 10% fetal bovine serum (FBS, Invitrogen), advanced DMEM/F12 (Invitrogen), 1% N2 neural supplement and without EGF.
All of the animals were handled according to the animal usage standards of the Association for Research in Vision and Ophthalmology (ARVO) and following approval by the Animal Research Committee of the Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine.

Wang Y., Zhang D., Tang Z., Zhang Y., Gao H., Ni N., Shen B., Sun H, & Gu P. (2018). REST, regulated by RA through miR-29a and the proteasome pathway, plays a crucial role in RPC proliferation and differentiation. Cell Death & Disease, 9(5), 444.

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Retinal Progenitor Cell Culture

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According to our previous studies, RPCs were obtained from fresh retinal tissue of postnatal day 1 C57BL/6 mice, and were cultured with proliferation medium containing advanced Dulbecco's modi ed Eagle's medium (DMEM)/F12 (Invitrogen, Carlsbad, CA, USA), 20 ng/ml recombinant epidermal growth factor (EGF, Invitrogen), 2 mM L-glutamine (Invitrogen) and 1% N2 neural supplement (Invitrogen) [9, 14] .
For differentiation study, RPCs were cultured with differentiation medium containing advanced DMEM/F12 (Invitrogen), 10% fetal bovine serum (FBS, Invitrogen), 1% N2 neural supplement (Invitrogen) and without EGF. All animal experiments were approved by the Animal Ethics Committee of the Second A liated Hospital, School of Medicine, Zhejiang University and were performed in compliance with the ARRIVE guidelines.

Ma J., Fang X., Chen M., Wang Y., & Zhang L. (2021). Hyaluronan-CD44 Interaction Regulates Mouse Retinal Progenitor Cells Migration, Proliferation and Neuronal Differentiation.

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Isolation and Culturing of Retinal Progenitor Cells

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RPCs were isolated from fresh retinal tissue of postnatal-day-one GFP transgenic C57BL/6 mice (a gift from Dr. Masaru Okabe, University of Osaka, Japan)27 (link)28 (link). The cells were seeded into flasks at a density of 2 × 10⁵ cells/ml with proliferation medium containing advanced DMEM/F12 (Invitrogen, Carlsbad, CA, USA), 1% N2 neural supplement (Invitrogen), 2 mM L-glutamine (Invitrogen), 100 U/ml penicillin-streptomycin (Invitrogen) and 20 ng/ml epidermal growth factor (EGF, Invitrogen)29 (link)30 (link). Every two or three days, the proliferation medium was changed, and the cells were passaged at regular intervals of three or four days. For differentiation, the cells were seeded at a density of 1 × 10⁵ cells/ml with differentiation medium, to which 10% fetal bovine serum (FBS) (Invitrogen) was added while EGF was removed; the cultures were then allowed to grow for 7 days. The culture medium was renewed every two days. All of the animals were handled according to the ARVO animal usage standards and following the approval by the animal care and use committee of the Schepens Eye Research Institute, where the original derivation of the cells was performed.

Ni N., Zhang D., Xie Q., Chen J., Wang Z., Deng Y., Wen X., Zhu M., Ji J., Fan X., Luo M, & Gu P. (2014). Effects of let-7b and TLX on the proliferation and differentiation of retinal progenitor cells in vitro. Scientific Reports, 4, 6671.

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Retinal Progenitor Cell Isolation and Culture

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Retinal progenitor cells (RPCs) were obtained from fresh retinal tissue of postnatal day 1 C57BL/6 mice. Next, the isolated RPCs were placed in a T25 flask and cultured using a proliferation medium containing advanced DMEM/F12 (Invitrogen, Carlsbad, CA, USA), 2 mM l-glutamine (Invitrogen), 1% N₂ neural supplement (Invitrogen), 20 ng/mL of epidermal growth factor (EGF, Invitrogen) and 100 U/mL of penicillin-streptomycin (Invitrogen). During the differentiation study, RPCs were incubated in differentiation medium with 10% fetal bovine serum (FBS, Invitrogen) without EGF.
All the animals were handled according to the Association for Research in Vision and Ophthalmology (ARVO)'s animal usage standards following authorization by the Animal Care and Use Committee of the Schepens Eye Research Institute, where the original source of cells was derived.

Sun N., Dou X., Tang Z., Zhang D., Ni N., Wang J., Gao H., Ju Y., Dai X., Zhao C., Gu P., Ji J, & Feng C. (2020). Bio-inspired chiral self-assemblies promoted neuronal differentiation of retinal progenitor cells through activation of metabolic pathway. Bioactive Materials, 6(4), 990-997.

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Schwann Cell Adhesion and Viability

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Etched glass coverslips were coated either with a 20 μg/mL solution of poly-D-lysine (PDL; Fisher) overnight at room temperature or 1 mg/mL iOA for 1 hour at 37°C. Neonatal rat Schwann cells (passage 2-3; Sciencell) were plated onto coated coverslips at 30 000 cells per coverslip (approximately 27 000 cells/cm²), allowed to adhere for 20 minutes at 37°C, and incubated in 3:1 DMEM:F12 medium containing 1% penicillin-streptomycin (Fisher), 2% N2 neural supplement (Invitrogen), 5 μM forskolin (Sigma), and 50 ng/mL neuregulin (R&D Systems). After 3 days, a live/dead assay was performed according to manufacturer’s instructions (Invitrogen). Fluorescence images were obtained at 10× magnification using an Olympus IX70 with DP80 dual color digital camera and CellSens software. The numbers of total cells and dead cells were counted using the “Analyze Particles” function in ImageJ (NIH). For total cell counts, individual cells were discriminated using the standard ImageJ “watershed” function in binary mode prior to analysis. Cell numbers were averaged for three frames in each sample (n=5, repeated three times).

Cornelison R.C., Gonzalez-Rothi E.J., Porvasnik S.L., Wellman S.M., Park J.H., Fuller D.D, & Schmidt C.E. (2018). Injectable Hydrogels of Optimized Acellular Nerve for Injection in the Injured Spinal Cord. Biomedical materials (Bristol, England), 13(3), 034110.

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Isolation and Expansion of Human Retinal Progenitor Cells

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hRPCs isolated from donated neural retinas of 16 to 18 weeks gestational age were obtained from the Eye Bank of He Eye Hospital in Shenyang, China. The donor signed the informed consent form. All works with human samples were performed after obtaining approval from the Institutional Review Board (IRB) of He Eye Hospital of He University (approval K007.01, March 2019). The protocol used a previously reported approach [13 (link)]. Briefly, the whole neuroretina was taken, then minced and digested with TrypLE express (Invitrogen) for 2 min. The cell pellet was resuspended using Ultraculture media (Lonza), supplemented with 1% N2 neural supplement, L-glutamine (2 mM), penicillin-streptomycin (100 U/ml), and recombinant human epidermal growth factor (EGF, 20 ng/ml) and basic fibroblast growth factor (bFGF, 20 ng/ml) (all purchased from Invitrogen), followed by inoculation in fibronectin pre-coated cell flasks in an incubator (37°C, 5% CO₂). The hRPCs were passaged when the confluence reached 80%. The hRPCs at passage 6 were used for the experiments.

Wang Z., Gao F., Zhang M., Zheng Y., Zhang F., Xu L., Cao L, & He W. (2020). Intravitreal Injection of Human Retinal Progenitor Cells for Treatment of Retinal Degeneration. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 26, e921184-1-e921184-10.

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Retinal Cell Isolation and Differentiation

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RPCs were harvested from fresh retinal tissue of postnatal-day-one GFP transgenic C57BL/6 mice (a gift from Dr. Masaru Okabe, University of Osaka, Japan). The cells were cultured with proliferation medium containing advanced Dulbecco′s Modified Eagle Media: Nutrient Mixture F-12 (DMEM/F12) (Invitrogen, Carlsbad, CA, USA), 1% N2 neural supplement (Invitrogen), 2 mM L-glutamine (Invitrogen), 100 U/ml penicillin-streptomycin (Invitrogen) and 20 ng/ml epidermal growth factor (EGF, Invitrogen) [44 (link)]. The culture medium was replaced every 2 days, and the cells were passaged at regular intervals of 3 or 4 days. For differentiation, the cells were cultured in differentiation medium containing 10% fetal bovine serum (FBS) (Invitrogen) without EGF. The cultures were then allowed to grow for seven days. The culture medium was renewed three times a week.
All animals were handled according to the ARVO animal usage standards and following approval by the animal care and use committee of the Schepens Eye Research Institute, where the original derivation of the cells was performed.

Zhang Y., Shen B., Zhang D., Wang Y., Tang Z., Ni N., Jin X., Luo M., Sun H, & Gu P. (2017). miR-29a regulates the proliferation and differentiation of retinal progenitors by targeting Rbm8a. Oncotarget, 8(19), 31993-32008.

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Retinal Progenitor Cell Isolation and Differentiation

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RPCs were separated from the fresh retinal tissue of postnatal-day-1 GFP-transgenic C57BL/6 mice (a gift from Dr Masaru Okabe, University of Osaka, Japan). The cells were cultured in T25 flasks with proliferation medium consisting of advanced DMEM/F12 (Invitrogen, Carlsbad, CA, USA), 1% N2 neural supplement (Invitrogen), 2 mM L-glutamine (Invitrogen), 100 U/ml penicillin-streptomycin (Invitrogen) and 20 ng/ml epidermal growth factor (recombinant human EGF, Invitrogen)46 (link). Half of the proliferation medium was changed every 2 days, and the clones were passaged through mechanical isolation or enzymatic trypsinisation at regular 3-day intervals. For RPC differentiation, the cells were trypsinised and seeded at a density of 1 × 10⁵ cells/ml with differentiation medium consisting of 10% foetal bovine serum (FBS) (Invitrogen) without EGF. Half of the culture medium was changed every 2 days. The cells were cultured at 37 °C and 5% CO₂. All animals were handled according to ARVO animal usage standards, following approval by the animal care and use committee of the Schepens Eye Research Institute, where the original derivation of the cells was performed.

Zhang D., Ni N., Chen J., Yao Q., Shen B., Zhang Y., Zhu M., Wang Z., Ruan J., Wang J., Mo X., Shi W., Ji J., Fan X, & Gu P. (2015). Electrospun SF/PLCL nanofibrous membrane: a potential scaffold for retinal progenitor cell proliferation and differentiation. Scientific Reports, 5, 14326.

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