Neurocult media

Manufactured by STEMCELL

Sourced in Canada

Neurocult media is a culture medium designed for the maintenance and expansion of neural stem and progenitor cells. It provides a defined, serum-free environment to support the growth of these cell types.

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11 protocols using neurocult media

Isolation and Culture of Primary Tumor Cells

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Primary cells were isolated directly from patient’s sample after surgery at Fondazione IRCCS Ca’ Granda-Ospedale Maggiore Policlinico, Milan, Italy. Written informed consent was obtained from all subjects involved in the study and the protocol was approved by a local Ethic Committee (IRB#275/2013).
Samples obtained from tumor cores (n=51) and/or non-neoplastic tumor margins (MG, n=20) were disaggregated enzymatically and mechanically using a tumor dissociation kit (Miltenyi Biotech, Bergisch Gladbach, Germany). Then, tumor cells were washed twice with HBSS (Thermo Fisher Scientific, Waltham, MA USA) and seeded in Neurocult media supplemented with NS-A Proliferation Kit (Human) and growth factors (bFGF and EGF; all from Stem Cell, Vancouver, BC, Canada) to maintain undifferentiated state, or RPMI supplemented with 10% FBS (both from Gibco-Thermo Fisher Scientific) for differentiated condition. MG cultures were seeded in RPMI supplemented with 10% FBS and maintained for up to one month in culture.

Bertolini I., Storaci A.M., Terrasi A., Di Cristofori A., Locatelli M., Caroli M., Ferrero S., Altieri D.C, & Vaira V. (2020). Interplay between V-ATPase G1 and small EV-miRNAs modulates ERK1/2 activation in GBM stem cells and non-neoplastic milieu. Molecular cancer research : MCR, 18(11), 1744-1754.

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Purification and Transplantation of Medulloblastoma Tumor Cells

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Cerebellar stem/progenitor cells (Prominin1+ cells) were purified by fluorescence activated cell sorting (FACS) from the cerebella of postnatal day 5–7 (P5–P7) C57BL/6J pups or MHC-I, TNFR1, TNFR2, TNFR1/2, LTβR or p53 knockout pups as previously described ^{11 (link),14 (link)}. To generate MP or MG tumors, cells were infected with viruses encoding Myc and either DNp53 (for MP tumors) or Gfi1 (for MG tumors). After overnight infection, cells were washed and 10,000 cells were stereotaxically injected into the cerebellum of 6- to 8-week-old NSG or Albino B6 (aB6) mice. Animals were monitored weekly using in vivo bioluminescence imaging and euthanized when they showed signs of MB. Tumors were then dissociated and resuspended in NeuroCult medium with proliferation supplement (STEMCELL Technologies) for subsequent experiments.
Tumor cells from conditional Ptch1 knockout (Math1-CreER^T2; Ptch1^flox/flox) mice, a model for SHH-driven medulloblastoma, were generated as previously described ^{19 (link)}. 0.5 to 1 million cells from frozen tumor stocks were stereotaxically implanted into the cerebellum of NSG mice. When animals showed signs of MB, tumors were harvested, resuspended in NeuroCult media with proliferation supplement (STEMCELL Technologies), and used for further experiments.

Garancher A., Suzuki H., Haricharan S., Chau L.Q., Masihi M.B., Rusert J.M., Norris P.S., Carrette F., Romero M.M., Morrissy S.A., Skowron P., Cavalli F.M., Farooq H., Ramaswamy V., Jones S.J., Moore R.A., Mungall A.J., Ma Y., Thiessen N., Li Y., Morcavallo A., Qi L., Kogiso M., Du Y., Baxter P., Henderson J.J., Crawford J.R., Levy M.L., Olson J.M., Cho Y.J., Deshpande A.J., Li X.N., Chesler L., Marra M.A., Wajant H., Becher O.J., Bradley L.M., Ware C.F., Taylor M.D, & Wechsler-Reya R.J. (2020). Tumor Necrosis Factor Overcomes Immune Evasion in p53-Mutant Medulloblastoma. Nature neuroscience, 23(7), 842-853.

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Generating Brain Tumor Cell Lines

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To generate BSG cell lines, tumors were isolated from symptomatic mice (described above) and enzymatically digested in Earl’s Balanced salt solution containing 4.7mg papain (Worthington) and 60μg /mL DNAse (Sigma Aldrich). Digestion was inactivated with ovomucoid (0.7 mg/mL) (Worthington) containing 14μg/mL DNAse. Cells were consecutively washed, triturated, and strained to obtain a single cell suspension. The cells were cultured in neurosphere media [Neurocult media (Stem Cell Technologies) supplemented with 5 ml cell proliferation supplement (Stem Cell Technologies), 500 μL penicillin and streptomycin, 10 μL human basic FGF (20 ng/ml), 5 μL human EGF (10 ng/ml), 50 μL heparin] and incubated at 37°C and 5% CO_2.

Hennika T., Hu G., Olaciregui N.G., Barton K.L., Ehteda A., Chitranjan A., Chang C., Gifford A.J., Tsoli M., Ziegler D.S., Carcaboso A.M, & Becher O.J. (2017). Pre-Clinical Study of Panobinostat in Xenograft and Genetically Engineered Murine Diffuse Intrinsic Pontine Glioma Models. PLoS ONE, 12(1), e0169485.

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Isolation and Culture of Embryonic and Adult Neural Stem Cells

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Embryonic neural stem cells (NSC) were isolated from either E14.5 embryos or P6 pups according to previous protocol [26 (link)]. Briefly, the meninges were removed and lateral and medial ganglionic eminences were harvested, mechanically dissociated in mouse NeuroCult media (Stem cell Technology). Cells were seeded in NeuroCult media enriched by FGF 10 ng/ml and EGF 20 ng/ml on low adhesion plates (Corning) at 100,000 cells/ml. After 3d, established neurospheres were passaged every 10d by mechanical dissociation. Adult NSCs were isolated from 5-month-old mice by precise dissection of the SVZ from a coronal section under a dissecting microscope. Adult NSCs were extracted, plated and passaged as described above.

Maire C.L., Ramkissoon S., Hayashi M., Haidar S., Ramkissoon L., DiTomaso E, & Ligon K.L. (2014). Pten Loss in Olig2 Expressing Neural Progenitor Cells and Oligodendrocytes Leads to Interneuron Dysplasia and Leukodystrophy. Stem cells (Dayton, Ohio), 32(1), 313-326.

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Murine DIPG Neurosphere Culture

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To generate murine DIPG neurospheres, whole tumors were extracted and incubated with 5mL papain solution (Papain 4.6 mg; EDTA 0.9 mg; Cysteine 0.9 mg; 5 mL Earls Balanced Salt Solution) plus 30μL DNase (10mg/mL, Sigma Aldrich) with trituration, followed by 2 mL of Ovomucoid solution (Ovomucoid stock solution: Ovomucoid protein 10 mg; 20 μL DNase) and centrifuged at 1100rpm. The pellet was then triturated in 0.5mL of ovomucoid, brought up with 5mL of Neurocult media (Stem Cell Technologies, #05700), centrifuged at 600rpm, and repeated 3 times. The cell pellet and aggregate supernatant were filtered and plated at a density of 500,000 cells per 25 cm² flasks in serum free neurosphere media (per 50 mL total volume: 44.5 mL Neurocult basal media (Stem Cell Technologies); 10% proliferation supplement (Stem Cell Technologies #05701); Pen-strep 1% (Invitrogen #15140–122); Human basic FGF, 20ng/mL (Invitrogen #13256–029); Human EGF, 10ng/mL (Invitrogen #PHG0314); Heparin, 2μg/mL (Stem Cell Technologies #07980). Cells were incubated at 37° C and split as required approximately once a week with Accutase (Stem Cell Technologies).

Halvorson K.G., Barton K.L., Schroeder K., Misuraca K.L., Hoeman C., Chung A., Crabtree D.M., Cordero F.J., Singh R., Spasojevic I., Berlow N., Pal R, & Becher O.J. (2015). A High-Throughput In Vitro Drug Screen in a Genetically Engineered Mouse Model of Diffuse Intrinsic Pontine Glioma Identifies BMS-754807 as a Promising Therapeutic Agent. PLoS ONE, 10(3), e0118926.

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Propagation and Characterization of Medulloblastoma Models

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The medulloblastoma cell line HD-MB03 (obtained from Till Milde, Hopp Children’s Cancer Center, Heidelberg) ^{18 (link)}, was cultured in RPMI-1640 medium (Gibco-ThermoFisher), supplemented with 10% fetal bovine serum (Seradigm), 100 units/mL penicillin, and 100 μg/mL streptomycin (Invitrogen), 0.1mM non-essential amino acids (Invitrogen).
Primary MB samples and PDX lines used for this study are listed in Supplemental Table 2 and in the Life Sciences Reporting Summary. Primary MB samples were collected during surgery at Rady Children’s Hospital (San Diego, CA). PDX lines were generated by implanting patient cells directly into the cerebellum of NSG mice, and propagating them from mouse to mouse without in vitro passaging; the identity and subgroup of each line was validated by gene expression and/or methylation analysis. For all experiments, cells were isolated from tumor-bearing mice, resuspended in NeuroCult media with proliferation supplement (STEMCELL Technologies), and assayed.

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Culturing Glioma Cell Lines

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The human glioma cell line U87 (or U-87MG) was purchased from ATCC and the U87EGFRvIII isogenic GBM cell line was obtained as described previously (Wang et al., 2006 (link)). U87EGFRvIII, U251, T98, U373 and A172 GBM cell lines were cultured in Dulbecco's Modified Eagle Media (DMEM, Cellgro), and PC9 cells were cultured in RPMI1640 (Gibco), supplemented with 10% fetal bovine serum (FBS, Hyclone) and 1% penicillin/streptomycin/glutamine (Invitrogen) in a humidified 5% CO2 incubator at 37°C. MDA-MB-361 cells were cultured in Leibovitz's L-15 media (Cellgro) supplemented with 20% FBS in a humidified 0% CO2 incubator at 37°C. Normal human astrocytes were purchased from Lonza and cultured per manufacturer's guidelines. GBM39, GBM6, HK301, GSC11, and GSC23 were cultured in Neurocult media (Stemcell Technologies) supplemented with EGF (Sigma), FGF (Sigma) and heparin (Sigma) in a humidified 5% CO2 incubator at 37°C.

Villa G.R., Hulce J.J., Zanca C., Bi J., Ikegami S., Cahill G.L., Gu Y., Lum K.M., Masui K., Yang H., Rong X., Hong C., Turner K.M., Liu F., Hon G.C., Jenkins D., Martini M., Armando A.M., Quehenberger O., Cloughesy T.F., Furnari F.B., Cavenee W.K., Tontonoz P., Gahman T.C., Shiau A.K., Cravatt B.F, & Mischel P.S. (2016). An LXR-Cholesterol Axis Creates a Metabolic Co-Dependency for Brain Cancers. Cancer cell, 30(5), 683-693.

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Establishment and Characterization of Human Glioblastoma Cell Lines

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Human glioblastoma cell lines A1207, LN340, LN18, LN428, LN382, LN464, U373, U87, LN443, U138, U118, SF767, LN319, LN215, U343, LN229, U178, LN235 and T98G were cultured in DMEM containing 10% FBS and 1% Penicillin-Streptomycin (Invitrogen, Carlsbad, CA) at 37°C in a 5% CO₂ incubator and passaged with Trypsin. NHA were purchased from ScienCell (Carlsbad, CA). NHA were passaged using Astrocyte Medium from ScienCell. The BT99, BT74, BT112, BT147, BT179, BT145 neurosphere line was provided by Dr. Keith Ligon (Dana Farber Cancer Institute). Neurosphere lines were cultured in NeuroCult media (StemCell Technologies, Vancouver, BC, Canada) supplemented with EGF, FGF and Heparin per manufacturer's instructions and grown at 37°C in a 5% CO₂ incubator. Stable cell lines were established by transfecting LN340 cells with the pCMV-miR-603 miRNA expression vector or empty vector control (SC400580, Origene, Rockville,MD). Stable cells were selected using neomycin. Single clones were isolated and cultured in DMEM containing 10% FBS and 1% Penicillin-Streptomycin (Invitrogen) at 37°C in a 5% CO₂ incubator.

Kushwaha D., Ramakrishnan V., Ng K., Steed T., Nguyen T., Futalan D., Akers J.C., Sarkaria J., Jiang T., Chowdhury D., Carter B.S, & Chen C.C. (2014). A genome-wide miRNA screen revealed miR-603 as a MGMT-regulating miRNA in glioblastomas. Oncotarget, 5(12), 4026-4039.

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Establishment and Characterization of GBM BTICs

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BTICs (n = 15) were derived from newly diagnosed and recurrent high-grade gliomas were obtained from the laboratory of Dr. Samuel Weiss at the University of Calgary during the period between 2008 and 2012. All BTIC lines were used within 25–30 passages of line establishment from primary cells. In brief, following informed consent from GBM patients, GBM BTICs were cultured from tumor specimens obtained during operative procedures as previously described [27 (link)] and approved by the University of Calgary Ethics Review Board and the Health Research Ethics board of Alberta—Cancer Commitee (HREBA, Protocol # HREBA.CC-16-0153). Newly diagnosed lines were derived from treatment-naïve patients and recurrent ones from patients who had been treated previously with TMZ and RT. BTICs were maintained at 37°C in a humidified 5% CO₂ environment and grown in serum-free NeuroCult^™ media (Stem Cell Technologies) supplemented with 0.1% heparin (Stem Cell Technologies), 0.1% rhesus fibroblast growth factor and 0.1% rhesus epidermal growth factor (Peprotech). Pyrosequencing to assess MGMT methylation status was performed by EpigenDx (Orlando, FL) using protocol ADS1552.

Yuan A.L., Ricks C.B., Bohm A.K., Lun X., Maxwell L., Safdar S., Bukhari S., Gerber A., Sayeed W., Bering E.A., Pedersen H., Chan J.A., Shen Y., Marra M., Kaplan D.R., Mason W., Goodman L.D., Ezhilarasan R., Kaufmann A.B., Cabral M., Robbins S.M., Senger D.L., Cahill D.P., Sulman E.P., Cairncross J.G, & Blough M.D. (2018). ABT-888 restores sensitivity in temozolomide resistant glioma cells and xenografts. PLoS ONE, 13(8), e0202860.

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Culturing Neural Stem Cells from Single-Cell Suspension

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After trypsinization, single-cell suspensions were obtained by filtering the cells using a 40 µm Cell Strainer (BD Biosciences). Then, cells were counted and 500 cells were grown in NeuroCult complete media consisting of NeuroCult Neural Stem Cell (NSC) Basal medium, 1/10 NeuroCult NSC Proliferation supplements, 20 ng/ml EGF, 10 ng/ml bFGF, and 2 μg/ml Heparin. NeuroCult media, supplements, and growth factors were all purchased from Stem Cell Technologies (Vancouver, BC, Canada). Cells were cultured for two weeks before the neurosphere containing more than twenty cells were counted.

Alshareef A., Gupta N., Zhang H.F., Wu C., Haque M, & Lai R. (2017). High expression of β-catenin contributes to the crizotinib resistant phenotype in the stem-like cell population in neuroblastoma. Scientific Reports, 7, 16863.

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