L glutamax

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L-GlutaMAX is a specialized laboratory product offered by Thermo Fisher Scientific. It is a stable, L-glutamine supplement designed for cell culture applications. The core function of L-GlutaMAX is to provide a readily available source of L-glutamine to support cellular growth and metabolism.

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L-Glutamine (Glutamax 100x) DMEM GlutaMAX™-l L-15 Leibovitz-glutamax L-glutamine (GlutaMAX™-I) DMEM) Glutamax 4.5 g/l glucose L-glutamine substitute (GlutaMAX™; L-gluthamine (Glutamax GlutaMAX L-glutamine supplement Dulbecco’s modified Eagle’s high glucose w/Glutamax (4.5 g/L) cell culture medium L-glutamax-1

143 protocols using l glutamax

Cell Culture Protocols for Various Cell Types

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HEK293 and HeLa cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMED; Gibco) supplemented with 10% FBS (Hyclone) and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin; Gibco). For MEFs, the cell culture medium consists of 10% FBS (Hyclone), 1 mM sodium pyruvate (Gibco), 0.055 mM β-mercaptoethanol (Gibco), 2 mM L-GlutaMax (Gibco), 0.1 mM NEAA (Gibco), and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin; Gibco). For iPSC generation cell culture media, 1000 U/ml LIF (Millipore) were added based on MEF media. iPSC stable clones were maintained on mitomycin C-treated MEF feeder cells in knockout DMEM medium (Gibco) supplemented with 15% KSR, 1 mM sodium pyruvate (Gibco), 0.055 mM β-mercaptoethanol (Gibco), 2 mM L-GlutaMax (Gibco), 0.1 mM NEAA (Gibco), 100 U/ml penicillin (Gibco), 100 μg/ml streptomycin (Gibco) and 1000 U/ml LIF (Millipore). CGR8 ES cells were cultured on gelatin-coated dishes, in GMEM medium supplemented with 10% FBS (Millipore), 1 mM sodium pyruvate (Gibco), 0.055 mM β-mercaptoethanol (Gibco), 2 mM L-GlutaMax (Gibco), 0.1 mM NEAA (Gibco), 100 U/ml penicillin (Gibco), 100 μg/ml streptomycin (Gibco) and 1000 U/ml LIF (Millipore). P19 EC cells were maintained on gelatin-coated dishes, in α-MEM medium supplemented with 10% FBS (Hyclone) and 100 U/ml penicillin (Gibco), 100 μg/ml streptomycin (Gibco).

Jin W., Wang L., Zhu F., Tan W., Lin W., Chen D., Sun Q, & Xia Z. (2016). Critical POU domain residues confer Oct4 uniqueness in somatic cell reprogramming. Scientific Reports, 6, 20818.

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Targeted USP14 Knockdown and PGJ2 Treatment

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The dicer siRNA substrates targeting the USP14 gene obtained from IDT (Coralville, Iowa), were encapsulated into lipid nanoparticles using microfluidic technology by Precision NanoSystems (SUB9KITS™; Vancouver, Canada). Cells were treated with 0.1 μg/ml of Neuro9™ RNAi nanoparticles as described in [68 (link)] for 72 h prior to treatment with 10 μM PGJ2 for 16 h. During siRNA treatment, cells remained in Neurobasal media supplemented with 2% B27 and 0.5 mM L-Glutamax (all from Invitrogen) at 37°C in a humidified atmosphere containing 5% CO₂. Following the 72 h incubation period with siRNA, the medium was changed to DMEM without serum, supplemented with 0.5 mM L-Glutamax and 1 mM sodium pyruvate (all from Invitrogen) and cells were treated with 10 μM PGJ2 for 16h.

Kiprowska M.J., Stepanova A., Todaro D.R., Galkin A., Haas A., Wilson S.M, & Figueiredo-Pereira M.E. (2017). Neurotoxic mechanisms by which the USP14 inhibitor IU1 depletes ubiquitinated proteins and Tau in rat cerebral cortical neurons: relevance to Alzheimer’s disease. Biochimica et biophysica acta, 1863(6), 1157-1170.

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Isolation and Culture of Blood and Cancer Cells

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Peripheral blood mononuclear cells (PBMCs) were obtained from healthy volunteers and B cell lymphoma and B-ALL patients after informed consent on protocols approved by the Baylor College of Medicine Institutional Review Board (H-15152, H-27471, H-19384 and H-31970). Capan-1 (pancreatic cancer cell line) and DU145 (prostate cancer cell line) were obtained from the American Type Culture Collection (Rockville, MD). NALM6 (pre-B-ALL cell line) and Raji (Burkitt lymphoma cell line) were gifted by Dr. Maksim Mamonkin. Cells were maintained in a humidified atmosphere containing 5% carbon dioxide (CO₂) at 37 °C. Capan-1 and DU145 cells were maintained in Iscove’s Modified Dulbecco’s Medium (IMDM; Gibco BRL Life Technologies, Inc., Gaithersburg, MD) while NALM6 and Raji cells were maintained in RPMI-1640 media (GE Healthcare Life Sciences, Pittsburgh, PA). Capan-1 cells were grown in IMDM containing 20% heat-inactivated fetal bovine serum (FBS) (Hyclone, Waltham, MA) with 2 mM L-GlutaMAX (Gibco BRL Life Technologies, Inc.) while other cell lines were grown in their specific media containing 10% FBS with 2 mM L-GlutaMAX.

Torres Chavez A., McKenna M.K., Canestrari E., Dann C.T., Ramos C.A., Lulla P., Leen A.M., Vera J.F, & Watanabe N. (2019). Expanding CAR T cells in human platelet lysate renders T cells with in vivo longevity. Journal for Immunotherapy of Cancer, 7, 330.

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Trypan Blue Viability Assay for SAEC

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Human SAEC were provided by Dr. Brooke Mossman and were previously characterized by Hei and colleagues (Piao et al., 2005 (link)). SAEC were cultured in advanced RMPI 1640 (Life Technologies, Waltham, MA) media with supplementation of 10% (v/v) heat-inactivated or gamma-irradiated fetal bovine serum (FBS) (HyClone, Logan, UT), 1% L-glutaMAX (Gibco, Grand Island, NY), and 1% Penicillin-Streptomycin-Neomycin (PSN) (Gibco, Manassas, VA). All cells were maintained and propagated at 37 °C in a humidified 5% CO₂ incubator.
A trypan blue staining assay was used to determine the viability of SAEC treated with SWCNTs. SAEC were plated in 6-well plates at the density of 3 × 10⁵ per well with complete RPMI media for 24 h. Then, cells were exposed to different doses of SWCNTs (0.2, 2.0, 20.0, 50.0 μg/mL) suspended in exposure media, which contains advanced RPMI supplemented with 1% L-glutaMAX and 1% PSN for 24 h. Cells were washed with phosphate buffered saline (PBS) and stained with trypan blue dye (Gibco, Grand Island, NY). Live/dead (blue) cells were differentiated and counted under a light microscope with a hemocytometer. At least 200 cells were counted for each treatment (n = 3) and the entire experiment was repeated 3 times.

Chen H., Humes S.T., Robinson S.E., Loeb J.C., Sabaraya I.V., Saleh N.B., Khattri R.B., Merritt M.E., Martyniuk C.J., Lednicky J.A, & Sabo-Attwood T. (2019). Single-walled Carbon Nanotubes Repress Viral-Induced Defense Pathways through Oxidative Stress. Nanotoxicology, 13(9), 1176-1196.

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Lacrimal Gland Differentiation from hiPS Cells

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Multi zonal ocular cell differentiation approach developed by Li and others was adopted for lacrimal gland differentiation (Li et al., 2019 (link)). hiPS cells were seeded on 1% hESC qualified Matrigel-coated six well plates and maintained in eye field differentiation (ED) medium composed of (DMEM/F12 (Gibco, Thermo Fisher Scientific) and neurobasal medium (Gibco, Thermo Fisher Scientific) (1:1) supplemented with 2 mM L-GlutaMAX (Gibco, Thermo Fisher Scientific), 0.1 mM non-essential amino acids (Lonza), 0.1 mM monothioglycerol (FUJIFILM Wako Pure Chemical Corporation), and 1% N2 MAX supplement (R&D Systems). 2% Matrigel was added to ED medium for first 2 days of differentiation. After 7 days of culture, medium was replaced with the ocular cell differentiation (OD) medium composed of DMEM/F12 supplemented with 10% knockout serum replacement (Gibco, Thermo Fisher Scientific), 2 mM L-GlutaMAX, 0.1 mM NEAA, and 0.1 mM monothioglycerol. Organoids maintained in culture for up to 45 days with daily medium refreshments.

Asal M., Koçak G., Sarı V., Reçber T., Nemutlu E., Utine C.A, & Güven S. (2023). Development of lacrimal gland organoids from iPSC derived multizonal ocular cells. Frontiers in Cell and Developmental Biology, 10, 1058846.

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Cultivation of Induced Pluripotent Stem Cells

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The piPSCs constructed by Ma et al. (2018) (link) were used in this study. The piPSCs were grown on feeder cells [mouse embryonic fibroblasts (MEFs) treated by mitomycin-C] and maintained in the standard medium as follows (termed LBCS medium): DMEM (Hyclone, United States) supplemented with 15% FBS (VISTECH, VIS59216269, New Zealand), 0.1 mM NEAA (Gibco, United States), 1 mM L-glutaMAX (Gibco), 0.1 mM β-mercaptoethanol (Sigma-Aldrich, United States), 100 U/ml of penicillin, 100 μg/ml of streptomycin, 10 ng/ml of LIF (Sino Biological, China), 10 ng/ml of bFGF (Sino Biological), 3 μM CHIR99021 (MCE, United States), 2 μM SB431542 (Selleck, China), and 4 μg/ml of doxycycline (DOX, Sigma-Aldrich). The piPSCs were subcultured by using TrypLE^TM Select (Invitrogen, United States) at a 1:50 ratio every 5–6 days. HEK293T cells used in this study were cultured in DMEM supplemented with 10% FBS, 0.1 mM NEAA (Gibco, United States), 1 mM L-glutaMAX (Gibco), 0.1 mM β-mercaptoethanol (Sigma-Aldrich, United States), 100 U/ml of penicillin, and 100 μg/ml of streptomycin.

Yu S., Zhang R., Shen Q., Zhu Z., Zhang J., Wu X., Zhao W., Li N., Yang F., Wei H, & Hua J. (2021). ESRRB Facilitates the Conversion of Trophoblast-Like Stem Cells From Induced Pluripotent Stem Cells by Directly Regulating CDX2. Frontiers in Cell and Developmental Biology, 9, 712224.

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Corneal Endothelial Cell Differentiation from hPSCs

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The hPSCs (2.5 × 10⁵ cells) were seeded on 1% Matrigel-coated dishes and grew into 30% confluence after 4 days’ culture, then cultured in neural crest differentiation medium (NDM) consisting of DMEM/F12 (Gibco), 20% knockout serum replacement (KSR, Gibco), L-GlutaMAX (2 mM, Gibco), MEM nonessential amino acids (0.1 mM, Gibco), β-mercaptoethanol (0.1 mM, Gibco), basic fibroblast growth factor (4 ng/mL, bFGF, R&D Systems), and 1 μM retinoic acid (RA, Sigma). After 5 days of culture, the differentiated cells grew into approximately 80% confluence and the medium was replaced with corneal endothelial differentiation medium (CDM) consisting of DMEM/F12 (Gibco), 0.2% BSA, bFGF (8 ng/mL), PDGF-BB (10 μg/mL, R&D Systems), DKK-2 (10 μg/ml, R&D Systems), insulin-transferring-selenium (Gibco), 2 mM L-GlutaMAX (Gibco), NEAA (0.1 mM), ascorbic acid (50 μg/mL, Sigma), Heregulin β-1 (10 ng/mL, Peprotech), IGF-1 (200 ng/mL, Peprotech), 50 × B27 (Gibco), β-mercaptoethanol (0.01 mM, Sigma), Y27632 (10 μM, Sigma), and SB431542 (1 μM, Millipore). The NCCs were induced into CEPs in the following 3 days, and toward CECs within 14 days. All cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂ and the medium was changed every day.

Li Z., Duan H., Jia Y., Zhao C., Li W., Wang X., Gong Y., Dong C., Ma B., Dou S., Zhang B., Li D., Cao Y., Xie L., Zhou Q, & Shi W. (2022). Long-term corneal recovery by simultaneous delivery of hPSC-derived corneal endothelial precursors and nicotinamide. The Journal of Clinical Investigation, 132(1), e146658.

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Culturing Rat Embryonic Neurons

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Primary neurons from either the hippocampus or the cerebral cortex of E17 rat embryos were cultured as described previously (Frost et al. 2010 (link)). Briefly, hippocampi or small pieces of frontal cerebral cortex were cut into small pieces, digested with 0.25% trypsin-EDTA, and mechanically dissociated by gentle pipetting with a series of small-bore Pasteur pipettes. Cells were resuspended in Neurobasal medium (Invitrogen) supplemented with 5% B27 supplement, 5% penicillin-streptomycin, and 2 mM L-GlutaMAX (all from Invitrogen) and then cultured in 12- or 24-well plates pre-coated with poly-L-lysine at 37 °C and 5% CO₂ in a humidified atmosphere. Primary neurons were cultured for 10–14 days prior to treatment with various concentrations of tunicamycin or thapsigargin (Sigma-Aldrich), as described in each experiment.

Shakya M., Yildirim T, & Lindberg I. (2020). Increased expression and retention of the secretory chaperone proSAAS following cell stress. Cell Stress & Chaperones, 25(6), 929-941.

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Dissociated Hippocampal Neuron Culture Protocol

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The preparation of cultured dissociated hippocampal neurons was described previously27 (link)30 . Before dissection of hippocampi, neonatal rats were sacrificed by decapitation, in accordance to the procedures approved by the animal care committee of Université Laval. The dissociated cells were plated on poly-d-lysine-coated glass coverslips (12 or 18 mm) at a density of 4225 or 860 cells/mm² respectively. Growth media consisted of Neurobasal and B27 (50:1), supplemented with penicillin/streptomycin (50 U/mL; 50 μg/mL) and 0.5 mM L-GlutaMAX (Invitrogen). Fetal bovine serum (2%; Hyclone) was added at time of plating. After 5 days, half of the media was changed without serum and with Ara-C (5 μM; Sigma-Aldrich) to limit proliferation of non-neuronal cells. Twice a week thereon, half of the growth medium was replaced with serum- and Ara-C–free medium. The neurons were transfected at 11–14 days in vitro (DIV) with Lipofectamine 2000 (Invitrogen) as described previously27 (link). For Ca²⁺ imaging, plasmids encoding GCaMP6s21 (link) and RCaMP1.07 (gift of J. Nakai) were transfected 1 day prior to measurements. CaMKII was tagged with a monomeric GFP as described previously27 (link).

Lavoie-Cardinal F., Salesse C., Bergeron É., Meunier M, & De Koninck P. (2016). Gold nanoparticle-assisted all optical localized stimulation and monitoring of Ca2+ signaling in neurons. Scientific Reports, 6, 20619.

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Hematopoietic Differentiation of hESCs/iPSCs

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For cell differentiation, OP9 cells were plated onto gelatinized 100-mm dishes in α-MEM (Invitrogen) containing 10% fetal bovine serum (FBS) (BI), 2 mM L-glutamax (Invitrogen), and 50 μg/ml penicillin/streptomycin (Gibco). After formation of confluent cultures on day 4, half of the medium was changed and cells were cultured for an additional two to three days. Undifferentiated hESCs and iPSCs were harvested by mechanical dissociation into small clumps and added to OP9 cultures at a density of 1.5 × 10⁶/20 ml per 100-mm dish in α-MEM supplemented with 10% FBS, 100 μM monothioglycerol (MTG; Sigma), 100 μM vitamin C (Sigma), 10 ng/ml IL-3 (Peprotech), 50 ng/ml SCF (Peprotech), 50 ng/ml IL-6 (Peprotech). Co-cultures were incubated for up to 10 days with a half-medium change every other day. Cells were harvested on day 10, and single-cell suspension was prepared for clonogenic, flow-cytometric assays, and MACS for CD34 + cells.

Ou Z., Niu X., He W., Chen Y., Song B., Xian Y., Fan D., Tang D, & Sun X. (2016). The Combination of CRISPR/Cas9 and iPSC Technologies in the Gene Therapy of Human β-thalassemia in Mice. Scientific Reports, 6, 32463.

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