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Neurocult basal media

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Neurocult basal media is a cell culture medium designed to support the growth and maintenance of neural cells, including neurons and glial cells. It provides the essential nutrients and growth factors required for the optimal growth and differentiation of these cell types.

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Lab products found in correlation

Proliferation supplement, by STEMCELL (4 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions) 10cm flasks, by Corning (2 mentions) Collagenase xi, by Merck Group (2 mentions) Dispase, by STEMCELL (2 mentions) Epidermal growth factor (egf), by Thermo Fisher Scientific (2 mentions) Accutase, by STEMCELL (2 mentions) Heparin, by STEMCELL (2 mentions) Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (2 mentions) Tc10 automated cell counter, by Bio-Rad (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) Fibroblast growth factor (fgf), by Thermo Fisher Scientific (1 mentions) Human egf, by Thermo Fisher Scientific (1 mentions) Human fgf basic, by Thermo Fisher Scientific (1 mentions) Pen strep 1, by Thermo Fisher Scientific (1 mentions) Neurocult media, by STEMCELL (1 mentions) Dnase, by Merck Group (1 mentions)

6 protocols using neurocult basal media

1

Myenteric plexus neuron isolation

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Plp1::GFP mice aged 12-16 weeks were euthanized and their small intestine was removed from duodenum to terminal ileum. The longitudinal muscle-myenteric plexus (LMMP) layer, which contains myenteric ganglia, was carefully dissected from underlying tissue under a dissecting microscope in ice-cold PBS supplemented with 10% bovine serum albumin. After dissection, LMMP tissue was digested for 60 minutes at 37° C in dispase (250 μg/ml; STEMCELL Technologies, Vancouver, BC) and collagenase XI (1mg/ml; Sigma-Aldrich, St. Louis, MO). Following digestion, the cells were filtered via a 40 micron filter to ensure a single-cell suspension. Immediately after digestion and filtering, cells were counted and resuspended at a density of 105 cells/mL in a 1:1 mixture of DMEM (Thermo Fisher, Waltham, MA) and NeuroCult Basal Media (STEMCELL Technologies, Vancouver, BC) supplemented with 20 ng/mL FGF, 20 ng/mL IGF1, 2% B27 supplement, 1% N2 supplement, 50 mM b-mercaptoethanol, and 75 ng/mL retinoic acid. 105 cells/mL in a total volume of 10 mL were placed 10cm flasks (Corning Inc, Corning, NY) at 37° C and 5% CO2 for 7-10 days. Media was replaced on day 5 by centrifuging cells at 250g for 3 minutes followed by re-suspension in fresh media and return to the same flasks and incubator for 5 more days. Cells from both male and female mice were used and were cultured separately.
Guyer R.A., Stavely R., Robertson K., Bhave S., Mueller J.L., Picard N.M., Hotta R., Kaltschmidt J.A, & Goldstein A.M. (2023). Single-cell multiome sequencing clarifies enteric glial diversity and identifies an intraganglionic population poised for neurogenesis. Cell reports, 42(3), 112194.
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2

Modeling Metabolic Stress in NPCs

Cited 1 time
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AGS NPCs (Neuronascent, Gaithersburg, MD, USA) and mouse NPCs (gift of Song lab, Baltimore, MD) have been previously described (Drew et al., 2016 (link); Ma et al., 2009 (link)). They were grown under standard conditions at 37°C and 5% CO2 with NeuroCult basal media (STEMCELL, Vancouver, BC, CA) with EGF (50 ng/ml, PeproTech, Inc, Rocky Hill, NJ, USA), FGF (100 ng/ml, PeproTech, Inc), heparin (0.002%), and proliferation supplements (STEMCELL). Early passage cultures (P2) were expanded and frozen and thawed in batches for use in experiments. These cultures contain cells ubiquitously expressing the NPC marker, Nestin, and the proliferation marker, Ki-67 (Figure 1—figure supplement 1). For in vitro modeling of metabolic stress, cells were exposed to either: (i) 1% hypoxia in a specialized incubator (Nuaire, Plymouth, MN, USA) saturated with Nitrogen/5% CO2; (ii) hypothermia in standard incubators maintained at lower temperatures; and (iii) complex I inhibition with the addition of rotenone to cell media. For cell proliferation determination, wells were seeded in triplicate with 50,000 cells. On subsequent consecutive days, cells were detached with Accutase (STEMCELL) and counted by automated cytometry (Nanoentek, Waltham, MA, USA).
Singhal N.S., Bai M., Lee E.M., Luo S., Cook K.R, & Ma D.K. (2020). Cytoprotection by a naturally occurring variant of ATP5G1 in Arctic ground squirrel neural progenitor cells. eLife, 9, e55578.
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3

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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4

Myenteric plexus neuron isolation

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Plp1::GFP mice aged 12-16 weeks were euthanized and their small intestine was removed from duodenum to terminal ileum. The longitudinal muscle-myenteric plexus (LMMP) layer, which contains myenteric ganglia, was carefully dissected from underlying tissue under a dissecting microscope in ice-cold PBS supplemented with 10% bovine serum albumin. After dissection, LMMP tissue was digested for 60 minutes at 37° C in dispase (250 μg/ml; STEMCELL Technologies, Vancouver, BC) and collagenase XI (1mg/ml; Sigma-Aldrich, St. Louis, MO). Following digestion, the cells were filtered via a 40 micron filter to ensure a single-cell suspension. Immediately after digestion and filtering, cells were counted and resuspended at a density of 105 cells/mL in a 1:1 mixture of DMEM (Thermo Fisher, Waltham, MA) and NeuroCult Basal Media (STEMCELL Technologies, Vancouver, BC) supplemented with 20 ng/mL FGF, 20 ng/mL IGF1, 2% B27 supplement, 1% N2 supplement, 50 mM b-mercaptoethanol, and 75 ng/mL retinoic acid. 105 cells/mL in a total volume of 10 mL were placed 10cm flasks (Corning Inc, Corning, NY) at 37° C and 5% CO2 for 7-10 days. Media was replaced on day 5 by centrifuging cells at 250g for 3 minutes followed by re-suspension in fresh media and return to the same flasks and incubator for 5 more days. Cells from both male and female mice were used and were cultured separately.
Guan L., Wims L.A., Kane R.R., Smuckler M.B., Morrison S.L, & Hawthorne M.F. (1998). Homogeneous immunoconjugates for boron neutron-capture therapy: Design, synthesis, and preliminary characterization. Proceedings of the National Academy of Sciences of the United States of America, 95(22), 13206-13210.
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5

MP Tumor Expansion and Culturing

Cited 1 time
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The development and culturing of MP tumors have been previously described59 (link). MP tumors were in vivo expanded in vivo in 6–8 week old NOD-SCID cerebellum. Cells from the mouse cerebellum were harvested after the animal reached endpoint (i.e., clinical: any head swelling, animal is quiet, circling, 10% weight loss from pre-injection). On the same day as tumor processing, After purification, neural stem cells and tumor cells were maintained in vitro at 1 × 106/mL cells in Neurocult basal media supplemented with 10% Proliferation supplement (Stem cell technologies), Penicillin/Streptomycin 1% (Thermo Fisher Scientific), basic Fibroblast growth factors (bFGF, 25 ng/mL, PeproTech) and Epidermal growth factor (EGF, 25 ng/mL, PeproTech). Cells were counted automatically with TC10 automated cell counter (Biorad). 4-hydroxytamoxifen (4OHT, 1 M in DMSO) was used as stock solution for 4OHT experiments.
Kameda-Smith M.M., Zhu H., Luo E.C., Suk Y., Xella A., Yee B., Chokshi C., Xing S., Tan F., Fox R.G., Adile A.A., Bakhshinyan D., Brown K., Gwynne W.D., Subapanditha M., Miletic P., Picard D., Burns I., Moffat J., Paruch K., Fleming A., Hope K., Provias J.P., Remke M., Lu Y., Reya T., Venugopal C., Reimand J., Wechsler-Reya R.J., Yeo G.W, & Singh S.K. (2022). Characterization of an RNA binding protein interactome reveals a context-specific post-transcriptional landscape of MYC-amplified medulloblastoma. Nature Communications, 13, 7506.
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6

Culturing Mouse Glioma Stem Cells

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The development and culturing of MP tumors have been previously described 58 (link) . MP tumors were in vivo expanded in vivo in 6-8 week old NOD-SCID cerebellum. Cells from the mouse cerebellum were harvested after the animal reached endpoint. On the same day as tumor processing, After puri cation, neural stem cells and tumor cells were maintained in vitro at 1x10 6 /mL cells in Neurocult basal media supplemented with 10% Proliferation supplement (Stem cell technologies), Penicillin/Streptomycin 1% (Thermo Fisher Scienti c), basic Fibroblast growth factors (bFGF, 25 ng/mL, PeproTech) and Epidermal growth factor (EGF, 25 ng/mL, PeproTech). Cells were counted automatically with TC10 automated cell counter (Biorad). 4-hydroxytamoxifen (4OHT, 1M in DMSO) was used as stock solution for 4OHT experiments.
Kameda-Smith M., Zhu H.H., Luo E., Venugopal C., Xella A., Yee B.A., Xing S., Tan F.E., Fox R., Brown K.R., Adile A., Bakhshinyan D., Chokshi C., Gwynne W.D., Subapanditha M., Picard D., Burns I., Moffat J., Fleming A., Hope K.J., Provias J., Remke M., Lu Y., Reya T., Reimand J., Wechsler‐Reya R.J., Yeo G., & Singh S.K. (2022). Characterization of an RNA binding protein interactome reveals the targetable post-transcriptional landscape of MYC-amplified medulloblastoma.
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