SH-SY5Y, Hela, and human alpha-synuclein overexpressing SH-SY5Y cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Primary cortical neurons were cultured from Sprague-Dawley rat embryos at embryonic day 18 and maintained in Neurobasal medium (Invitrogen, Carlsbad, CA, USA) with L-glutamine and B-27 supplement (Invitrogen). Hela cells were transfected using lipofectamine 2000 (Invitorgen) according to the manufacturer's instruction. After 24 hr of transfection, the cells were used for further experiments. The plasmid for UCH-L1-myc was kindly provided by Prof. K. J. Lee at Ewha Womans University, Korea. UCH-L1 knockdown SH-SY5Y cells were prepared using lentiviral constructs that expressed shRNA for UCH-L1 (Sigma-Aldrich) as described previously [31 (link)] and selected using puromycin.
L glutamine and b 27 supplement
Manufactured by Thermo Fisher Scientific
Sourced in United States
L-glutamine is an amino acid that is often added to cell culture media to support cell growth and proliferation. The B-27 supplement is a proprietary serum-free supplement that is commonly used to supplement neural cell culture media to support the growth and differentiation of neuronal cells.
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5 protocols using l glutamine and b 27 supplement
1
Establishing Cell Models for Parkinson's Research
2
Primary Hippocampal Neuron Culture from Mouse Embryos
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WT (FVB/N stain) mouse embryos, at 18 days of gestation (from four WT females), were used to prepare primary hippocampal neuronal cultures as previously described [34 (link)]. Briefly, hippocampi were dissected in HBSS (without Ca2+ and Mg2+) supplemented with 1 mM sodium pyruvate and 10 mM HEPES and dissociated by trituration with a fire-polished Pasteur pipette. Supernatants were then centrifuged for 1 min at 1000×g. The pellets were resuspended in HBSS. The cell suspensions were plated at a density of 5 × 105/cm2 in neurobasal medium (Invitrogen) supplemented with 0.5 mM l -glutamine and B27 supplement (Invitrogen) and grown on glass coverlips pretreated with poly-d -lysine (Sigma-Aldrich).
3
Analyzing α-Synuclein Roles in Parkinson's
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WT and parkin KO mouse embryonic fibroblast (MEF) cells, kindly provided by Dr. Youle RJ (National Institutes of Health, Bethesda), and human α-synuclein overexpressing SH-SY5Y cells [36 , 37 ] were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum (FBS). Cells were transfected using lipofectamine 2000 or lipofectamine RNAiMax (Invitrogen, Carlsbad, CA) according to the manufacturer’s instruction. After 48 or 72 h of transfection, cells were used for Western blotting or imaging analysis. Primary cortical neurons were cultured from Sprague-Dawley rat embryos at embryonic day 18, and maintained in Neurobasal medium (Invitrogen, Carlsbad, CA) with L-glutamine and B-27 supplement (Invitrogen, Carlsbad, CA). All animal procedures used in the present study were conducted according to the guidelines established by the Ajou University School of Medicine Ethics Review Committee for Animal Experiments.
4
Culturing SH-SY5Y cells and primary neurons
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SH-SY5Y cells were grown in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS). Primary cortical neurons were cultured from Sprague-Dawley rat embryos or human A53T α-syn overexpressing transgenic (TG) mice at embryonic day 18, maintained in neurobasal medium (Invitrogen, Carlsbad, CA) with L-glutamine and B-27 supplement (Invitrogen, Carlsbad, CA).
5
Lentiviral Vector Construction for Caveolin-1 Mutants
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Plasmids for WT cav-1 and WT cav-1-EGFP were constructed using PCR. The plasmid for Y14A cav-1 was constructed using the Quick-Change site-directed mutagenesis kit (Stratagene, La Jolla, CA). Each construct was subcloned into pCDH-EF1 for generating lentiviral vectors. All plasmids were veri ed via DNA sequencing and prepared using the Maxi prep Kit (Qiagen, Valencia, CA).
Cell culture SH-SY5Y cells were grown in Dulbecco's modi ed Eagle's medium (DMEM), supplemented with 10% fetal bovine serum (FBS). Primary cortical neurons were cultured from Sprague-Dawley rat embryos or human A53T α-syn overexpressing transgenic (TG) mice at embryonic day 18, maintained in neurobasal medium (Invitrogen, Carlsbad, CA) with L-glutamine and B-27 supplement (Invitrogen, Carlsbad, CA).
Cell culture SH-SY5Y cells were grown in Dulbecco's modi ed Eagle's medium (DMEM), supplemented with 10% fetal bovine serum (FBS). Primary cortical neurons were cultured from Sprague-Dawley rat embryos or human A53T α-syn overexpressing transgenic (TG) mice at embryonic day 18, maintained in neurobasal medium (Invitrogen, Carlsbad, CA) with L-glutamine and B-27 supplement (Invitrogen, Carlsbad, CA).
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