Gm18453
Manufactured by Coriell Institute for Medical Research
Sourced in United States
GM18453 is a cell line derived from a skin biopsy sample. It is a fibroblast cell line established from a male donor. This cell line is available for research use from the Coriell Institute for Medical Research.
Automatically generated - may contain errors
Lab products found in correlation
Gm05659, by Coriell Institute for Medical Research (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Hygromycin, by Thermo Fisher Scientific (2 mentions)
Geneticin, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Rencell nsc maintenance media, by Merck Group (2 mentions)
Gm03123, by Coriell Institute for Medical Research (2 mentions)
Fugene 6 transfection reagent, by Roche (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Stemflex medium, by Thermo Fisher Scientific (2 mentions)
Matrigel, by Corning (2 mentions)
Polybrene, by Merck Group (1 mentions)
Epidermal growth factor (egf), by Merck Group (1 mentions)
293ft cell, by Thermo Fisher Scientific (1 mentions)
Basic fibroblast growth factor (bfgf), by Merck Group (1 mentions)
Gm00498, by Coriell Institute for Medical Research (1 mentions)
Neurobasal, by Thermo Fisher Scientific (1 mentions)
Cytosine d arabinofuranoside, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
10 protocols using gm18453
1
Fibroblasts and HeLa Cell Culture Protocols
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Fibroblast cell lines from a healthy male (GM05659, RRID: CVCL_7434) and a male patient with a homozygous mutation in NPC1I1061T (GM18453, RRID: CVCL_DA78, from the NIGMS repository) were purchased from the Coriell Institute. Fibroblasts were grown in Eagle’s Minimum Essential Medium (MEM) with Earle’s salts and non-essential amino acids (SIGMA, Cat # M5650) supplemented with 2 mM L-glutamine (GIBCO, Cat # 25030–081), 15% non-inactivated fetal bovine serum (GIBCO, Cat # 26140–079) and 0.2% penicillin/streptomycin (GIBCO, Cat # 15140–122), passaged twice a week, and incubated in 5% CO2 at 37°C.
HeLa cells stably expressing ABCA1-GFP were provided by Dr. Alan Remaley, NHLBI, Bethesda, MD. HeLa cells were grown in DMEM (GIBCO, Cat # 11995–065) supplemented with 10% non-inactivated fetal bovine serum and 0.2% penicillin/streptomycin, passaged twice a week, and incubated in 5% CO2 at 37°C. Expression of ABCA1-GFP HeLa cells was induced by adding 150 μg/ml geneticin (GIBCO, Cat # 10131–035) and 200 μg/ml Hygromycin (Invitrogen, Cat # 10687010).
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2
Generating Induced Neural Stem Cells from Fibroblasts
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Induced neural stem cells (iNSCs) were generated from normal donor skin fibroblasts (GM05659; Coriell Institute for Medical Research, Camden, NJ, USA) and NPC patient-derived human Dermal Fibroblasts (hDFs, GM03123 (NPC1P237S/I1061T), GM18453 (NPC1I1061T/I1061T); Coriell Institute for Medical Research). Viral production and transduction were performed as described previously. Briefly, retroviral pMX-SOX2 and pMX-HMGA2 were transfected into 293 FT cells along with VSV-G and gag/pol plasmids using Fugene 6 transfection reagent (Roche, Indianapolis, IN, USA). The viral supernatants were collected at 48- and 72-h post-transfection and used to infect hDFs with 5 μg/mL polybrene (Sigma-Aldrich, St. Louis, MO, USA). For neural stem cell induction, the medium was changed to NSC maintenance medium (ReNcell NSC maintenance media; Millipore, Billerica, MA, USA) with basic fibroblast growth factor (bFGF; Sigma-Aldrich, St. Louis, MO, USA) and epidermal growth factor (EGF; Sigma-Aldrich, St. Louis, MO, USA) after expansion of the infected cells. NSC-like colonies were picked and cultured in neurosphere culture conditions. To generate a homogenous population of iNSCs, cells were maintained as neurospheres and cultured as attached cells on PLO/FN-coated dishes, repeatedly. NPC-iNSC lines from NPC1 mutant human fibroblast were generated up to 10 independent clones.
3
Generation of iNSCs from Patient Fibroblasts
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The iNSCs were established from human NPC patient-derived skin fibroblasts (GM03123, GM18453; Coriell Institute for Medical Research, USA) and were characterized in a previous study [18 (link)]. The pMX-SOX2 and pMX-HMGA2 retroviral constructs were transfected into 293 FT cells (Invitrogen, USA) to produce high viral titers using FuGENE 6 Transfection Reagents (Roche Diagnostics, USA) and the viral supernatants were collected and used to infect NPC patient-derived fibroblasts. The transduced fibroblasts were cultured in NSC maintenance medium (ReNcell NSC Maintenance Media; Millipore, USA) with basic fibroblast growth factor (bFGF; Sigma, USA) and epidermal growth factor (EGF; Sigma) added to induce neural stem cells. NSC-like colonies were picked and cultured under neurosphere conditions as attached cells on poly-L-ornithine- and fibronectin-coated dishes repeatedly to generate homogenous iNSCs. As reported previously, the iNSCs showed an NSC-like morphology and expressed NSC-specific markers such as PAX6 and NESTIN. Furthermore, the iNSCs demonstrated differentiation into neurons, astrocytes, and oligodendrocytes, indicating that the generated iNSCs could function as NSCs.
4
Fibroblast Culture for NPC Research
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5
Fibroblasts and HeLa Cell Culture Protocols
6
Establishment of Diverse Cell Lines for Neurological Studies
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Fibroblast cell lines derived from an apparently healthy male (GM05659) and a male patient with the NPC1I1061T mutation (GM18453) were purchased from Coriell Institute. Additional fibroblast cell lines derived from patients, NPC1I1061T, I1061T and NPC1I1061T, P1007A, were generously provided by Dr. Forbes D. Porter (National Institutes of Health, Bethesda, MD). Fibroblasts were cultured in MEM supplemented with 2 mM L-glutamine, 15% non–heat-inactivated FBS, and 0.2% penicillin/streptomycin. Cells were passaged twice weekly and incubated in 5% CO2 at 37°C. PSEN+/+ (WT) and PSEN1/PSEN2 double-knockout (PSEN−/−) MEFs were a generous gift from Drs. David Kang, Angels Almenar, and Lawrence S.B. Goldstein (University of California, San Diego, San Diego, CA). WT, NPC1−/−, and SCAP−/− CHO cells were kindly provided by Daniel Ory (Washington University, St. Louis, MO). MEFs, CHO cells, and tsA201 cells were cultured in DMEM supplemented with 10% FBS and 0.2% penicillin/streptomycin. Hippocampal Neurons were isolated from rats at gestation day 18. Neurons were cultured in Neurobasal (21103-049; Gibco) supplemented with B27 (17504-044; Gibco), Glutamax (35050-061; Gibco), 5% FBS, and 0.2% penicillin/streptomycin. On DIV 7, cytosine-D-arabinofuranoside (251010; Millipore) at 1:1,000 was added to neuronal cultures to inhibit astrocyte growth.
7
Culturing and Maintaining HeLa and NPC1-deficient Fibroblasts
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HeLa cells were maintained at 37 °C in a humidified 5% CO2 atmosphere in DMEM with Glutamax supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (all from Life technologies). Human NPC1-deficient fibroblasts (GM18453) were obtained from the Coriell Institute and maintained at 37 °C in a humidified 5% CO2 atmosphere in MEM supplemented with 15% fetal bovine serum and 1% penicillin/streptomycin (all from Life technologies). Cells were negative for mycoplasma contamination.
8
NPC1 Fibroblast Homozygous Mutant Study
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Human NPC1 fibroblasts GM18453 (homozygous NPC1 mutant I1061T) were from Coriell Institute (Camden, NJ). All human skin fibroblasts were maintained in MEM supplemented with 10% FBS. For the inhibitor treatments, cells were maintained in MEM supplemented with 5.5% FBS and 20 mM Hepes.
9
Fibroblast-derived iPSC Culture
10
Fibroblast-derived iPSC Culture
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A patient fibroblast line (GM18453) was obtained from Coriell Cell Repositories and cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin and 100 μg/ml streptomycin in a humidified incubator with 5% CO2 at 37 °C. The iPSC line, TRNDi001-D, was cultured in StemFlex medium (Thermo Fisher Scientific) on Matrigel (Corning, 354277)-coated plates at 37 °C in humidified air with 5% CO2 and 5% O2. The cells were dissociated with Dulbecco’s Phosphate Buffered Saline (DPBS) containing 0.5 mM Ethylenediaminetetraacetic acid (EDTA) and passaged when they reached 70% confluency.
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