Sk n sh

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SK-N-SH is a neuroblastoma cell line derived from a bone metastasis of a neuroblastoma. It is a commonly used in vitro model for the study of neuroblastoma biology and the development of potential therapeutic agents.

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11 protocols using sk n sh

Parkinson's Disease In Vitro Cell Model

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Human neuroblastoma cell line SK-N-SH and mice dopaminergic neuronal cell line MN9D from Procell (Wuhan, China) were maintained in DMEM medium (Gibco, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS, Gibco) and 1% antibiotics (Gibco) in a moist incubator at 37°C under 5% CO₂.
For PD in vitro cell model, SK-N-SH and MN9D cells were stimulated with 1 mM MPP⁺ (Sigma) at 37°C for 24 h, respectively.

Xiao X., Tan Z., Jia M., Zhou X., Wu K., Ding Y, & Li W. (2021). Long Noncoding RNA SNHG1 Knockdown Ameliorates Apoptosis, Oxidative Stress and Inflammation in Models of Parkinson’s Disease by Inhibiting the miR-125b-5p/MAPK1 Axis. Neuropsychiatric Disease and Treatment, 17, 1153-1163.

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Differentiation of SK-N-SH cells into neuronal cells

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Human SK-N-SH cell line was obtained from American Type Culture Collection (ATCC #HTB-11). Cells were cultured in polystyrene tissue culture flasks (Corning) as a monolayer in Eagle’s Minimum Essential Medium (EMEM; ATCC). This medium was supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% penicillin-streptomycin-neomycin (Sigma). As this cell line is a mixture of different cells, retinoic acid (RA; Sigma) was used for differentiating SK-N-SH cells [23 (link)] into more neuronal cells [24 (link)]. Approximately 30,000 cells (per well in 6-well plates) were cutured in supplemented EMEM media for two days, followed by RA (10μM) treatment for two weeks and media was replaced every 3–4 days [23 (link), 25 (link)]. Using light microscopy, differentiated neuronal cultures were validated with the presence of neuronal cell markers (NeuN, PSD95 and NCAM) during the differentiation process [23 (link), 26 (link)] (Refer supplementary information).

Kaushik G., Xia Y., Pfau J.C, & Thomas M.A. (2017). Dysregulation of autism-associated synaptic proteins by psychoactive pharmaceuticals at environmental concentrations. Neuroscience letters, 661, 143-148.

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Human iPSC-derived Neural Progenitor Cells

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Human induced pluripotent stem cell (IPSC)-derived neural progenitor cells (NPCs) (Ax0015; Axol, Cambridge, United Kingdom) were cultured in neural maintenance basal medium with supplements (Ax0031; Axol) according to the manufacturer’s specification. Human IPSC-derived NPCs were grown on plates coated with 20 µg/ml laminin (L2020; Sigma-Aldrich). Human neuroblastoma SK-N-SH and human glioblastoma U87-MG cells were purchased from Sigma-Aldrich and grown in Eagle’s minimum essential medium (EMEM) with Earle’s balanced salt solution (EBSS [Lonza, Breda, The Netherlands) containing 10% heat-inactivated fetal bovine serum (HI-FBS [Lonza]), 100 U penicillin (Gibco Life Sciences, USA), 100 µg/ml streptomycin (Gibco), 2 mM l-glutamine (Lonza), 1% nonessential amino acids (Lonza), 1 mM sodium pyruvate (Gibco), and 1.5 mg/ml sodium bicarbonate (Lonza). Both immortalized cell lines SK-N-SH and U87-MG were used below passage 25. Vero cells (ATCC, USA) were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% HI-FBS, 100 µg/ml streptomycin, 100 U penicillin, 2 mM l-glutamine, 1% sodium bicarbonate, and 1% HEPES buffer (all from Gibco). Human NPCs are primary cells, while the other cells are from immortalized cell lines. All cells used in this study tested negative for Mycoplasma sp.

Anfasa F., Siegers J.Y., van der Kroeg M., Mumtaz N., Stalin Raj V., de Vrij F.M., Widagdo W., Gabriel G., Salinas S., Simonin Y., Reusken C., Kushner S.A., Koopmans M.P., Haagmans B., Martina B.E, & van Riel D. (2017). Phenotypic Differences between Asian and African Lineage Zika Viruses in Human Neural Progenitor Cells. mSphere, 2(4), e00292-17.

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Establishing PD Cell Model with MPP+ in Human Neuroblastoma Cells

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Human neuroblastoma cells of SK-N-SH and SK-N-BE were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in Dulbecco’s modified Eagle medium (DMEM; Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1 × penicillin & streptomycin (Gibco, the final concentration of 100 U/mL penicillin and 100 µg/mL streptomycin). SK-N-SH or SK-N-BE cells re-suspended in complete medium were incubated under a humid atmosphere of 5% CO₂ at 37 °C.
1-methyl-4-phenyl pyridinium (MPP⁺; Sigma-Aldrich, Louis, MO, USA) was used to establish PD cell model. To select the most appropriate concentration of MPP⁺, 0, 0.5, 1, 2, or 3 mM MPP⁺ were added into SK-N-SH and SK-N-BE cells, respectively. 2 mM was selected as the corresponding concentration for subsequent assays. Besides, SK-N-SH or SK-N-BE cells were pretreated with 100 µM MPP⁺ for 24 h to establish the in vitro cell model for PD.

Lv K., Liu Y., Zheng Y., Dai S., Yin P, & Miao H. (2021). Long non‐coding RNA MALAT1 regulates cell proliferation and apoptosis via miR-135b-5p/GPNMB axis in Parkinson’s disease cell model. Biological Research, 54, 10.

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Maintenance and Authentication of Cell Lines

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The human SHSY5Y, SKNSH, SKNAS and HEK293T cell lines were obtained from the American Type Culture Collection (respectively ATCC #CRL-2266, HTB-11, and #CRL-11268); the human IMR32 cell line was obtained from SIGMA (86041809). SHSY5Y, SKNAS, SKNSH, and HEK293T cell lines were grown in Dulbecco’s Modified Eagle Medium (DMEM; Sigma); IMR32 cell line was grown in Minimal Essential Eagle Medium (MEM; Sigma). The medium was supplemented with 10% heat-inactivated FBS (Sigma), 1 mmol/L L-glutamine, penicillin (100 U/mL), and streptomycin (100mg/mL;Invitrogen). The cells were cultured at 37° C, 5% CO2 in a humidified atmosphere. The cell lines used for all the experiments were reauthenticated and tested as mycoplasma-free. Early-passage cells were used and cumulative culture length was less than 3 months after resuscitation.

Cimmino F., Avitabile M., Diskin S.J., Vaksman Z., Pignataro P., Formicola D., Cardinale A., Testori A., Koster J., de Torres C., Devoto M., Maris J.M., Iolascon A, & Capasso M. (2018). Fine mapping of 2q35 high-risk neuroblastoma locus reveals independent functional risk variants and suggests full-length BARD1 as tumor-suppressor. International journal of cancer, 143(11), 2828-2837.

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Dose and Time-Dependent MPP+ Exposure in Neuroblastoma

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Human neuroblastoma cells SK-N-SH were commercially acquired from the American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained in Dulbecco's modified Eagle medium (DMEM; Gibco, Carlsbad, CA, USA) with 10 % fetal bovine serum (FBS; Gibco) at 37 °C with 5 % CO₂. Next, SK-N-SH cells were stimulated with different doses (0 mM, 0.25 mM, 0.5 mM and 1 mM) of MPP⁺(Sigma, St Louis, MO, USA) for 48 h or treated with 1 mM MPP⁺ for different times (0 h, 12 h, 24 h and 48 h).

Zhao J., Li H, & Chang N. (2020). LncRNA HOTAIR promotes MPP+-induced neuronal injury in Parkinson’s disease by regulating the miR-874-5p/ATG10 axis. EXCLI Journal, 19, 1141-1153.

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Culturing and Transfecting Cell Lines

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HEK293 (FreeStyle™ 293-F Cells, Invitrogen), Neuro2A (ATCC) and SK-N-SH (Sigma) cells were cultured in DMEM medium with 10% FBS and 1% Pen-Strep at 37 °C with 5% CO₂. SHSY5Y cells were cultured in DMEM/F12 medium with 15% FBS, 1% non-essential amino acids and 1% Pen-Strep at 37 °C with 5% CO₂. Transfection was performed using the Lipofectamine 2000 (Life technologies) and transfected cells were harvested 48 or 72 hours post-transfection. Stable cell lines were selected in the presence of 500 μg/ml G418 (Life Technologies).

Li Y., Collins M., Geiser R., Bakkar N., Riascos D, & Bowser R. (2015). RBM45 homo-oligomerization mediates association with ALS-linked proteins and stress granules. Scientific Reports, 5, 14262.

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Analyzing EB1 Comet Dynamics in Glioblastoma and Neuroblastoma Cells

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Cells were grown on 8-well chamber slides (Labtek, Thermo Scientific, Roskilde, Denmark), precoated for 1 hour with fibronectin (10 μg/ml) for U87-MG or with type I collagen (30µg/ml) for SK-N-SH (Sigma Aldrich), to be treated for 6 hours with MTAs and inhibitors. As previously described [32 (link)], cells were incubated with the anti-EB1 (clone 5; BD Biosciences, San Jose, CA) and α-tubulin (clone DM1A; Sigma Aldrich) primary antibodies, and then with Alexa488 or 568-conjugated secondary antibodies (Molecular Probes). Staining was observed using either a Leica DM-IRBE microscope or a Leica TCS SP5 confocal laser-scanning microscope (Leica, Heidelberg, Germany). Images were acquired using Metamoph software or the Leica Confocal software, and were processed using Image J software. For each experimental condition, at least 400 EB1 comets (in 40 cells) were examined to measure their length.

Grand M.L., Rovini A., Bourgarel-Rey V., Honore S., Bastonero S., Braguer D, & Carre M. (2014). ROS-mediated EB1 phosphorylation through Akt/GSK3β pathway: implication in cancer cell response to microtubule-targeting agents. Oncotarget, 5(10), 3408-3423.

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Differentiating SK-N-SH Cells into Neuron-Like Cells

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Human SK-N-SH cell line was obtained from American Type Culture Collection (ATCC #HTB-11). Cells were cultured in Eagle’s Minimum Essential Medium (EMEM; ATCC). This media was supplemented with 1 % penicillin-streptomycin-neomycin (Sigma) and 10 % (v/v) fetal bovine serum (FBS). Retinoic acid (RA; Sigma) was used to induce SK-N-SH cells [12 (link)] to differentiate into more neuron-like cells [13 (link)] because this cell line is a mixture of different cell types. Approximately 15,000 cells were cultured in T-75 flask (Corning) supplemented with EMEM media for two days, and then Retinoic acid (10 μM) was added. Cells were treated with RA for two weeks and media was replaced every three-four days [12 (link)]. Cultures were monitored visually using light microscopy for morphological changes, and evaluated for neuronal cell markers (NeuN, PSD95 and NCAM) during the differentiation process [12 (link), 14 (link)].

Kaushik G., Xia Y., Yang L, & Thomas M.A. (2016). Psychoactive pharmaceuticals at environmental concentrations induce in vitro gene expression associated with neurological disorders. BMC Genomics, 17(Suppl 3), 435.

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Cell Culture and Transfection Protocols for Multiple Cell Lines

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HeLa, HEK293, SK-N-SH, RT4-D6P2T (from Sigma, 93011415), and LAMP1/2 double knock-out or wild-type MEFs (gifts from Dr. P. Saftig, Kiel, Germany) were cultured in DMEM (D6046) medium with 10% FBS (12133C) (Sigma). mRFP-GFP-LC3–stably expressing HeLa cells, described previously (28 (link), 60 (link)), were cultured in DMEM with 10% FBS. Human primary schwannomas were cultured as described previously (63 (link)) with full ethical approval.
Plasmids were transfected into cells either alone or with siRNA using Lipofectamine 2000 (Thermo Scientific), according to the manufacturer's instructions, for the length of time indicated in each figure legend.

Button R.W., Roberts S.L., Willis T.L., Hanemann C.O, & Luo S. (2017). Accumulation of autophagosomes confers cytotoxicity. The Journal of Biological Chemistry, 292(33), 13599-13614.

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