Cells were suspended in the sphere induction medium, which is based on neural stem cell medium. The basal medium for the sphere induction medium is DMEM/F12 (Sigma-Aldrich, Tokyo, Japan) supplemented with 10 mM HEPES (Sigma-Aldrich), 1× antibiotic-antimycotic solution (Sigma-Aldrich), 0.6% glucose (Sigma-Aldrich), 1 mg/mL transferrin, 250 μg/mL insulin (Sigma-Aldrich), 0.6 mM putrescine (Sigma-Aldrich), 0.3 μM sodium selenite (Sigma-Aldrich), and 0.2 μM progesterone (Sigma-Aldrich). Complete sphere induction medium was prepared by adding 2 μg/mL heparin (Sigma-Aldrich), 10 ng/mL human recombinant EGF (Sigma-Aldrich), 10 ng/mL bFGF (Merck Millipore, Tokyo, Japan), 10 ng/mL leukemia inhibitory factor (LIF, Merck Millipore), 60 μg/mL N-acetyl-L-cysteine (NAC, Sigma-Aldrich), and 1/50 vol. neural survival factor-1 (NSF-1, Lonza, Tokyo, Japan) to the basal medium. Briefly, cells were collected and washed to remove serum and then cultured in the sphere induction medium at 37°C in a humidified atmosphere of 5% CO2 in air. The next day, induction was begun, and floating cells were transferred into a hydrophilic ultra low attachment flask (Corning, Corning, NY).
Nsf 1
Manufactured by Lonza
Sourced in Japan
The NSF-1 is a compact, high-precision laboratory equipment designed for a range of analytical and research applications. It features a robust construction and advanced functionality to deliver accurate and reliable results. The core function of the NSF-1 is to provide a controlled environment for precise measurements and experimentation.
Automatically generated - may contain errors
Lab products found in correlation
Basic fibroblast growth factor (bfgf), by Merck Group (4 mentions)
Putrescine, by Merck Group (3 mentions)
B27 supplement, by Thermo Fisher Scientific (3 mentions)
Insulin, by Merck Group (2 mentions)
Heparin, by Merck Group (2 mentions)
Hepes, by Merck Group (2 mentions)
Sodium selenite, by Merck Group (2 mentions)
Transferrin, by Merck Group (2 mentions)
Progesterone, by Merck Group (2 mentions)
Antibiotic antimycotic solution, by Merck Group (2 mentions)
Epidermal growth factor (egf), by Merck Group (2 mentions)
N acetyl l cysteine, by Merck Group (2 mentions)
Recombinant human egf, by Merck Group (1 mentions)
Dmem f12, by Merck Group (1 mentions)
N acetyl l cysteine nac, by Merck Group (1 mentions)
Ultra low attachment flask, by Corning (1 mentions)
Leukemia inhibitory factor lif, by Merck Group (1 mentions)
N2 supplement, by Thermo Fisher Scientific (1 mentions)
Epidermal growth factor egf, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
8 protocols using nsf 1
1
Sphere Induction of Neural Progenitors
2
Isolation and Characterization of Human Fetal Neural Progenitor Cells
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Human NPCs were isolated from human fetal brains provided by the University of Washington Medical Center Laboratory of Developmental Biology (R24HD000836-51). Informed consent was obtained from all tissue donors and all clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki. The UNMC Scientific Research Oversight Committee (SROC) and Institutional Review Boards at the University of Washington Medical Center and UNMC approved the protocol. Cells were isolated from human fetal brains as previously described (Nunes et al., 2003 (link)). Briefly, single cell suspensions were cultivated in NS-A medium (STEMCELL, Mountain View, Canada), containing N2 supplement (1:100; Fisher Scientific, Waltham, MA, USA), neural survival factor-1 (NSF1; 1:50; Lonza, Walkersville, MD, USA), LIF (10 ng/ml; Sigma-Aldrich, St. Louis, MO, USA), epidermal growth factor (EGF; 20 ng/ml; Fisher, MA, USA) and basic fibroblast growth factor (bFGF; 20 ng/ml; STEMCELL), for 14 days before passage. Four hours before injection, the neurospheres were dissociated into single cell suspension with 0.25% Trypsin (Gibco, Grand Island, NY, USA). The single cell suspensions were plated on polystyrene-coated coverslips and cultured for 48 h in vitro. Immunofluorescent cytology was applied to investigate the differential potential of the NPCs.
3
Culturing Hippocampal Neurons from Rat
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Eight 35 mm cell culture dish each containing four collagen-coated coverslips (Iwaki) were initially coated with 30 μg/mL poly-L-lysine filter sterilized in borate buffer in a 35 mm cell culture dish at 37°C. After incubation for an hour, poly-L-lysine/borate buffer was removed from the dishes. 1.5 mL of PNGM media supplemented with NSF-1 (Lonza) was added to the dish to wash out residual poly-L-lysine. Before final plating of hippocampal neurons, this media was removed. A tube of rat hippocampal neurons (Lonza R-Hi-501) were thawed in a 37°C water bath for about 2 min and transferred to a 50 mL conical tube. While stirring the tube gently, 16 mL of NSF-1/PNGM media was slowly added to the cells to prevent osmotic shock. After all the media was added, 2 mL of cells were added to each plate containing coverslips. After incubating in 37°C for about 4 h, 1.5 mL of media was removed from each plate and 2 mL of fresh NSF-1/PNGM media was added. Cells were incubated at 37°C and 5% CO2 for 10–14 days before imaging. To maintain the cells, 0.75 mL of media was removed and 1 mL of NSF-1/PNGM was added once or twice a week while neurons were growing. Neurons were used after a minimum of 10 days and no longer than 4 weeks of culture.
4
Serum-free Neurosphere Culture and Laminin-based Differentiation
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Cells were initially cultured in serum-free medium which is based on neural stem cell medium. The basal medium for the sphere induction is DMEM-F12 supplemented with 10 mM HEPES (Sigma-Aldrich), 1× antibiotic antimycotic solution (Sigma-Aldrich), 0.6% glucose (Sigma-Aldrich), 1 mg/ml transferrin, 250 μg/ml insulin (Sigma-Aldrich), 0.6 mM putrescine (Sigma-Aldrich), 0.3 μM sodium selenite (Sigma-Aldrich), and 0.2 μM progesterone (Sigma-Aldrich). Complete sphere induction medium was prepared by adding 2 μg/ml heparin (Sigma-Aldrich), 20 ng/ml EGF (Sigma-Aldrich), 20 ng/ml bFGF (Merck Millipore, Tokyo, Japan), 10 ng/ml LIF (Merck Millipore), 1/50 Vol NSF-1 (Lonza, Tokyo, Japan), and 60 μg/ml N-acetyl-L-cysteine (Sigma-Aldrich). Upon the formation of spheres, the sphere cells (YPK2-Sp and YPK5-Sp) were collected. YPK2-Sp or YPK5-Sp were then transferred to a laminin-coated dish with the sphere culture medium containing 20 μl/ml B27 supplement (Life Technologies), 1× antibiotic antimycotic solution, 75 μg/ml BSA (Sigma-Aldrich), 10 ng/ml EGF, and 10 ng/ml bFGF. Medium was renewed by a 50% change every 7 days. Cells became attached and gradually divided and increased in number (YPK2-Lm and YPK5-Lm).
5
Isolation and Culture of Cancer Stem-like Cells
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Cancer stem‐like cell‐enriched populations were obtained from YPK and SW480 cells as previously described.11 In brief, cells were first cultured in serum‐free medium containing LIF (Merck Millipore, Darmstadt, Germany), NSF‐1 (Lonza, Tokyo, Japan), and NAC (Sigma‐Aldrich Japan) to induce tumor spheres. The obtained spheres were collected and transferred to laminin‐coated dishes with sphere culture medium containing B27 supplement (Life Technologies), epidermal growth factor (Sigma‐Aldrich Japan), and basic fibroblast growth factor (Merck Millipore); half of the medium was changed every week. The resultant cells, designated YPK2‐Lm, YPK5‐Lm, and SW480‐Lm, gradually attached to the substratum and grew for 1–2 months; they were used to identify molecules differentially expressed in P‐CSLCs and parental cells by proteomics.
6
Isolation and Culture of Rat DRG Neurons
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The lumbar (L3-L5) DRG SNs were isolated as described 15 (link). Primary rat DRG neuronal cells (Lonza, R-DRG-505, Alpharetta, GA) were cultured in primary neuron growth medium (Lonza, #CC-4461) with 2% FBS, L-glutamine, gentamycin sulfate-amphotericin (GA-1000, Lonza, #CC-4083) and neural survival factor-1 (NSF-1, Lonza, #CC-4323) as instructed. Immortalized rat DRG neuronal cells, 50B11, were a generous gift from Dr. Hoke, Johns Hopkins University and cultured in Neurobasal medium (Gibco) supplemented with 10% FBS, 1 x B27 supplement (Gibco), 0.2% glucose and 0.5 mM glutamine (neuron growth medium) 30 (link). The rat DRG/mouse neuroblastoma hybrid cells, F11, were cultured as described 53 (link).
7
Culturing Neuronal Cell Lines and DRG Neurons
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SK-N-BE (2) (ATCC® CRL-2271™) and SH-SY5Y (ECACC 94030304) cell lines were grown in Dulbecco's Modified Eagle Medium (DMEM)/F-12 medium supplemented with 10% fetal bovine serum, 1% penicillin–streptomycin, 1% l -glutamine and 25 mm HEPES. HEK293T/17 cells (ATCC® CRL-11268™) were grown in DMEM high-glucose (4.5 g/l) medium supplemented with 10% fetal bovine serum, 1% penicillin–streptomycin, 1% l -glutamine, 1% sodium pyruvate. DRG neurons were prepared from neonatal (P3–P4) wild type mice, by following the protocol described in (51 ,64 (link)), and plated onto coverslips coated with 30 μg/ml poly-d -lysine (Sigma-Aldrich) and 2 μg/ml laminin (Thermofisher). Dissected neurons were maintained on coverslips in primary neuron basal medium (PNBM, Lonza) supplemented with 1% l -glutamine (Lonza), 0.1% gentamicin sulfate/amphotericin-B (Lonza), 2% NSF-1 (Lonza) and 50 ng/ml of mouse NGF. Twenty-four hours after seeding, 2.5 μm cytosine β-d-arabinoside (AraC, Sigma) was added for inhibition of glia proliferation. Neuronal culture medium was changed every 3–4 days, removing about 1/3 of the volume and substituting it with warm, fresh neuron growth medium.
8
Sphere Induction Medium for Neural Stem Cells
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Cells were suspended in the sphere inducing medium, which was based on a neural stem cell medium [16 (link)]. This medium, used to induce floating sphere cells, was DMEM/Nutrient Mixture F-12 Ham supplemented with 0.6% glucose, 10 mM HEPES, 2 μg/mL heparin, 0.1 mg/mL transferrin, 25 μg/mL insulin, 60 μM putrescine, 30 nM sodium selenite, 20 nM progesterone, 10 ng/mL human recombinant epidermal growth factor (all from Sigma-Aldrich Japan, Tokyo, Japan), 10 ng/mL basic fibroblast growth factor (Merck Millipore, Tokyo, Japan), 10 ng/mL leukemia inhibitory factor (Merck Millipore), 60 μg/mL N-acetyl-L-cysteine (Sigma-Aldrich), and 1/50 volume NSF-1 (Lonza, Tokyo, Japan).
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