Glutamaxtm 1 supplement

Manufactured by Thermo Fisher Scientific

Sourced in United States

GlutaMAX™-I Supplement is a cell culture medium supplement developed by Thermo Fisher Scientific. Its core function is to provide a stable source of L-glutamine, an essential amino acid required for cell growth and proliferation in cell culture applications.

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7 protocols using glutamaxtm 1 supplement

Primary Hippocampal Neuron Culture from Rat Pups

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Primary hippocampal neuron culture.
Rat (Sprague Dawley) pups at the postnatal 0-day (P0) were sacrificed to harvest CA1 and CA3 of the hippocampal regions of the brain tissue as previously described (23, 40) . All procedures were performed according to the animal protocol approved by the Department of Health, Government of Hong Kong. Around 2 × 10 4 neurons were plated on each Poly D-Lysine (P7886, Sigma-Aldrich, USA)-coated cover glass with a (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
diameter of 12 mm (Glaswarenfabrik Karl Hecht GmbH & Co KG, Germany) in 24well plates. Two days after plating the neurons, 10 µM uridine (U3003, Sigma-Aldrich) and 10 µM 5-fluorodeoxyuridine (F0503, Sigma-Aldrich) were added to inhibit the proliferation of glial cells in cultured neurons (41) . The neurons were cultured in the Neurobasal TM -A Medium (10888022, Thermo Fisher Scientific, USA) supplemented with 2.5 % FBS, 1 % Penicillin-Streptomycin (15140122, Thermo Fisher Scientific), 500 µM GlutaMAX TM -I Supplement (A1286001, Thermo Fisher Scientific), and 2 % B-27 TM Supplement (17504001, Thermo Fisher Scientific). Cultured neurons were maintained in a 5% CO2 incubator at 37°C before experiments were performed.

Park C., Chen X., Tian C., Park G.N., Chenouard N., Lee H., Yeo X.Y., Jung S., Bi G., Tsien R.W., & Park H. (2020). Inhibitory synaptic vesicles have unique dynamics and exocytosis properties.

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Neuroprotective Effects of Honokiol and Magnolol

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Honokiol and magnolol were purchased from Nacalai Tesque Inc. (Kyoto, Japan). Rat adrenal medulla PC12 cells were obtained from American Type Culture Collection (Manassas, VA, USA). 6-OHDA and RPMI-1640 medium were purchased from Sigma-Aldrich (St Louis, MO, USA). Antibiotic-Antimycotic (100 ×) liquid, horse serum and GlutaMAX TM -I Supplement were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Fetal bovine serum was purchased from Valley Biomedical (Winchester, VA, USA). The CellTiter-Glo ® Luminescent Cell Viability Assay kit was obtained from Promega Co. (Madison, WI, USA). Hydrogen peroxide was purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Cell culture Petri dishes (collagen type I coated ware) and 24-well cell culture multidisc (collagen type I coated ware) were from AGC Techno Glass Co. Ltd. (Shizuoka, Japan). The Bio-Rad Protein Assay was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). All other reagents were of the highest grade commercially available.

Nishiyama T., Masuda Y., Izawa T., Ohnuma T., Ogura K, & Hiratsuka A. (2019). Magnolol protects PC12 cells from hydrogen peroxide or 6-hydroxydopamine induced cytotoxicity. The Journal of toxicological sciences, 44(11).

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Isolation and Culture of Primary Rat Hippocampal Neurons

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Primary rat hippocampal neuron (PRHN; A104841-01) was purchased from Gibco™, USA. PRHN was isolated from day-18 Fischer 344 rat embryos, then cultured using the Neurobasal®-medium (Gibco™, 21103049) supplemented with GlutaMAXTM-I Supplement (Gibco™, 35050061) and B-27® Serum Free Supplement (Gibco™, 17504044) with proportions based on the protocol provided by the manufacturer. In addition, L-glutamine (Gibco™, 25030081) was supplemented for the first 4 days of culturing in accord with the manufacturer’s suggested protocol. The PRHN was plated with a neuronal density of 900 cells/mm² which have a maturity rate that suits our experiment design (45 (link)). PRHN was cultured on a culture plate coated with 0.01% poly-L-lysine (Sigma-Aldrich, 25988-63-0): molecular weight-70000–150000 according to the coating method described by Todd et al. (10 (link)). The cells were cultured in incubator at a temperature of 36 °C–38 °C with 5% CO₂. The culture was maintained, aspirating half of the media from each well and replacing it with fresh composite media every third day.

Chong P.N., Sangu M., Huat T.J., Reza F., Begum T., Yusoff A.A., Jaafar H, & Abdullah J.M. (2018). Trkb-IP3 Pathway Mediating Neuroprotection in Rat Hippocampal Neuronal Cell Culture Following Induction of Kainic Acid. The Malaysian Journal of Medical Sciences : MJMS, 25(6), 28-45.

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Culturing Human and Rat Stem Cells

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Human bone marrow-mesenchymal stem cells (hBM-MSCs) were purchased from Cell Engineering For Origin (CEFO) (Korea) and cultured in T75 flasks (Falcon; Corning, USA) according to the supplier’s recommendations. hBM-MSCs were cultivated in mesenchymal stem cell growth medium (Gibco, Grand Island, NY, USA) as previously described (Jeong and Cho, 2015 (link)). Passage seven (P-7) hBM-MSCs were used. Rat fetal neural stem cells (NSCs) were purchased from Gibco (Grand Island, NY, USA) and cultured in T75 flasks (Falcon) according to the supplier's recommendations. NSCs were cultivated in growth medium containing 2% StemPro^® NSC supplement (Gibco, Grand Island, NY, USA), 2 mM GlutaMax^TM I supplement (Gibco, Grand Island, NY, USA), 20 ng/ml basic fibroblast growth factor (bFGF) (Gibco, Carlsbad, CA, USA), and 20 ng/ml epidermal growth factor (Gibco, Carlsbad, CA, USA) in KnockOut^TM Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY, USA). P-3 NSCs were used for the experiments. Human osteosarcoma cells (U2OS) were cultured in DMEM/F12 (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (FBS) (Origin Canada; Gibco, Grand Island, NY, USA), 2 mM L-glutamine, and 1% penicillin/streptomycin antibiotics at 37°C in a 5% CO₂ incubator.

Jeong S.G., Ohn T., Jang C.H., Vijayakumar K, & Cho G.W. (2020). The Role of Stress Granules in the Neuronal Differentiation of Stem Cells. Molecules and Cells, 43(10), 848-855.

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Derivation of Human Neural Stem Cells

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Human neural stem cell line derived from the National Institutes of Health (NIH) approved H9 human embryonic stem cells (H9-hNSCs) were purchased from GIBCO, USA. The H9-hNSCs were seeded at a density of 1.0 × 10⁵ cells/cm² in a CELLstart^TM CTS^TM (GIBCO, USA) coated T25 flask added with 3 mL of complete StemPro NSC SFM culture medium comprised of 1X Knockout^TM D-MEM/F-12 (GIBCO, USA), 2 mM GlutaMAX^TM–I Supplement (GIBCO, USA), 20 ng/mL basic Fibroblast Growth Factor (GIBCO, USA), 20 ng/mL Epidermal Growth Factor (GIBCO, USA) and 2% StemPro Neural Supplement (GIBCO, USA). The cells were allowed to adhere for 24 h in a CO₂ incubator at 37 °C in the presence of 5% CO₂. The medium was replaced with equal volume of pre-warmed complete StemPro NSC SFM every two days and cultured until confluence prior to subsequent preconditioning treatments.

Kang I.N., Lee C.Y, & Tan S.C. (2019). Selection of best reference genes for qRT-PCR analysis of human neural stem cells preconditioned with hypoxia or baicalein-enriched fraction extracted from Oroxylum indicum medicinal plant. Heliyon, 5(7), e02156.

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Neural Stem Cell Culture Protocol

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The NSs medium contained 2% StemPro® Neural Supplement (Gibco) and 1% B27 supplements (Invitrogen); recombinant human fibroblast growth factor (FGF)-basic (bFGF, 20 ng/ml), recombinant human epidermal growth factor (EGF, 20 ng/ml), GlutaMAXTM-I Supplement (1%, Gibco), in Dulbecco’s modified Eagle’s medium (DMEM)/ F12 (1:1) containing 10% fetal bovine serum (FBS, Gibco) and 1% antibiotics.

Zhang X., Jiang W., Xie C., Wu X., Ren Q., Wang F., Shen X., Hong Y., Wu H., Liao Y., Zhang Y., Liang R., Sun W., Gu Y., Zhang T., Chen Y., Wei W., Zhang S., Zou W, & Ouyang H. (2022). Msx1+ stem cells recruited by bioactive tissue engineering graft for bone regeneration. Nature Communications, 13, 5211.

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Culturing Human Fetal Neural Stem Cells

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The hNSCs (StemPro^TM Neural Stem Cells) are cryopreserved human fetal brain-derived neural stem cells derived from cortex of a male fetus donor aged 16 weeks that were purchased from Gibco, United States (Cat. no. A15654). It was cultured in T25 flasks (BD Falcon), or in 6-well plates (BD Falcon) with KnockOut^TM DMEM/F-12 (Gibco, United States, cat. no. 12660012) supplemented with 2% StemPro^TM Neural Supplement (Gibco, Cat. no. A1050801), 20 ng/mL of fibroblast growth factor (FGF) basic recombinant human, 20 ng/mL epidermal growth factor (EGF) recombinant human and 2 mM GlutaMAX^TM-I Supplement (Cat. no. 35050), 6 U/mL heparin (Sigma, Cat. no. H3149), and 200 μM ascorbic acid (Sigma, Cat. no. A8960). For adhesion, Geltrex^® (Gibco, Cat. no. A14133) was used to coat the 6-well plate. The hNSCs were cultured at 37°C in a 95% air/5% CO2 humidified incubator. The medium was changed every 2 days.

Achour M., Ferdousi F., Sasaki K, & Isoda H. (2021). Luteolin Modulates Neural Stem Cells Fate Determination: In vitro Study on Human Neural Stem Cells, and in vivo Study on LPS-Induced Depression Mice Model. Frontiers in Cell and Developmental Biology, 9, 753279.

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