Neurocult ns a proliferation media

Manufactured by STEMCELL

Sourced in Canada

NeuroCult NS-A proliferation media is a cell culture media designed to support the proliferation of neural stem and progenitor cells in vitro.

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Lab products found in correlation

Heparin, by STEMCELL (4 mentions) Laminin, by Merck Group (3 mentions) Rhegf, by STEMCELL (3 mentions) Basic fibroblast growth factor (bfgf), by STEMCELL (3 mentions) Poly l ornithine, by Merck Group (3 mentions) Mycoalert mycoplasma detection kit, by Lonza (3 mentions) Basic fibroblast growth factor (bfgf), by Abcam (1 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Epidermal growth factor (egf), by Thermo Fisher Scientific (1 mentions) Epidermal growth factor (egf), by Miltenyi Biotec (1 mentions) Dmem media, by Thermo Fisher Scientific (1 mentions) Accutase, by STEMCELL (1 mentions) Fibroblast growth factor (fgf), by Miltenyi Biotec (1 mentions) Phenol red, by Wisent (1 mentions) Fetal bovine serum (fbs), by Wisent (1 mentions) L glutamine, by Wisent (1 mentions) Np 40, by Merck Group (1 mentions) Fugw vector, by Addgene (1 mentions) Trc shrna library, by Merck Group (1 mentions)

7 protocols using neurocult ns a proliferation media

Differentiation of MSCs into NPCs

Cited 1 time

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MSCs at passage 3 (3 biological replicates) were differentiated into NPCs as previously described [19 (link)]. Briefly, MSCs (collected from three SD rats) at P3 were plated in three (triplicate) ultra-low attachment plates at a density of 1 × 10⁶ cells/mL and were induced into NPCs with NeuroCult® NS-A proliferation media (STEMCELL Technologies, Vancouver, BC, Canada) supplemented with 20 ng/ml of bFGF (C/N: 4039–10; BioVision, CA, USA), 20 ng/ml of EGF (C/N: PMG8045; Gibco, Life Technologies, Carlsbad, CA, USA) and 1% penicillin–streptomycin (Gibco, Life Technologies, Carlsbad, CA, USA). Cells were monitored daily, and growth factors were supplemented every other day.

Khan A.A., Huat T.J., Al Mutery A., El-Serafi A.T., Kacem H.H., Abdallah S.H., Reza M.F., Abdullah J.M, & Jaafar H. (2020). Significant transcriptomic changes are associated with differentiation of bone marrow-derived mesenchymal stem cells into neural progenitor-like cells in the presence of bFGF and EGF. Cell & Bioscience, 10, 126.

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Culturing Glioblastoma Cell Lines

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A172, T98G, and U87MG glioblastoma cell lines were purchased from ATCC. Cells were cultured in DMEM media (ThermoScientific) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (P/S, ThermoScientific) according to ATCC instructions. Glioma neurosphere cell lines BT112, BT228, and BT333 were obtained from the DFCI Center for Patient Derived Models (CPDM) under a material transfer agreement and maintained as described previously (Touat et al 2020 Nature). Briefly, cells were grown in Neurocult NS-A Proliferation Media (StemCell) supplemented with 0.0002% heparin (StemCell Technologies), EGF (20 ng/ml), and FGF (10 ng/ml; Miltenyi) in a humidified atmosphere of 5% CO2 at 37 °C on low-attachment plates (62 (link)) and were dissociated with Accutase (StemCell Technologies) for passaging and plating.

Giglio R.M., Hou N., Wyatt A., Hong J., Shi L., Vaikunthan M., Fuchs H., Nima J.P., Malinowski S.W., Ligon K.L., McFaline-Figueroa J.R., Yosef N., Azizi E, & McFaline-Figueroa J.L. (2024). A heterogeneous pharmaco-transcriptomic landscape induced by targeting a single oncogenic kinase. bioRxiv.

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Primary HGG Cell Line Culture Protocols

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We thank Drs. Angel M. Carcaboso and Dinesh Rakheja for generously sharing primary HGG-derived cell lines. The HSJD-GBM002 cell line was cultured in NeuroCult NS-A proliferation media (StemCell Technologies) supplemented with bFGF (10 ng/mL), rhEGF (20 ng/mL), and heparin (0.0002%) (StemCell Technologies) and on plates coated with poly-L-ornithine (0.01%) (Sigma) and laminin (0.01 mg/mL) (Sigma). KNS-42, PS10-801 and CMC1118G8 cell lines were cultured in DMEM containing 4.5 g/L glucose, L-glutamine, phenol red, and 10% FBS (Wisent). All lines were tested monthly for mycoplasma contamination (MycoAlert Mycoplasma Detection kit by Lonza), and STR fingerprinting was regularly performed. Clinical information (age, sex) and mutation status of primary HGG cell lines are presented in Table S7.

Chen C.C., Deshmukh S., Jessa S., Hadjadj D., Lisi V., Andrade A.F., Faury D., Jawhar W., Dali R., Suzuki H., Pathania M., A D., Dubois F., Woodward E., Hébert S., Coutelier M., Karamchandani J., Albrecht S., Brandner S., De Jay N., Gayden T., Bajic A., Harutyunyan A.S., Marchione D.M., Mikael L.G., Juretic N., Zeinieh M., Russo C., Maestro N., Bassenden A.V., Hauser P., Virga J., Bognar L., Klekner A., Zapotocky M., Vicha A., Krskova L., Vanova K., Zamecnik J., Sumerauer D., Ekert P.G., Ziegler D.S., Ellezam B., Filbin M.G., Blanchette M., Hansford J.R., Khuong-Quang D.A., Berghuis A.M., Weil A.G., Garcia B.A., Garzia L., Mack S.C., Beroukhim R., Ligon K.L., Taylor M.D., Bandopadhayay P., Kramm C., Pfister S.M., Korshunov A., Sturm D., Jones D.T., Salomoni P., Kleinman C.L, & Jabado N. (2020). Histone H3.3G34-mutant interneuron progenitors co-opt PDGFRA for gliomagenesis. Cell, 183(6), 1617-1633.e22.

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Evaluating mAb-Conjugated Protein Expression

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GSCs were seeded at 250,000 cells/well in poly(l-ornithine)-, poly-L-ornithine (PLO)- (Sigma P4957), and laminin (Thermo CB-40232)-coated 6-well plates and allowed to adhere overnight in Neurocult NS-A proliferation media (Stem Cell 05751). mAb conjugates (or AONs or mAb alone) were added to 500 μL Opti-MEM media and added to the cells for a final Opti-MEM:Neurocult ratio of 1.5:0.5 v/v and mAb-AON concentration of 150 nM. 24 hr later, an additional 1 mL Neurocult media was added. After a total of 72 hr of incubation at 37°C, cells were collected and lysed using 0.1% NP-40 (Sigma 74385). Protein expression was assessed via western blot analysis of DRR with α-tubulin as a loading control.

Arnold A.E., Malek-Adamian E., Le P.U., Meng A., Martínez-Montero S., Petrecca K., Damha M.J, & Shoichet M.S. (2018). Antibody-Antisense Oligonucleotide Conjugate Downregulates a Key Gene in Glioblastoma Stem Cells. Molecular Therapy. Nucleic Acids, 11, 518-527.

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Astrocyte-Derived Glioma Stem Cells Knockdown

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All cell lines used in this study were tested for mycoplasma contamination every three months at the MSKCC Antibody and Bioresource Core. Parental immortalized normal human astrocytes were a gift from R.O. Peiper (UCSF)^{61 (link)}. TS 543, TS 603, and 08-0537 are patient-derived GSCs^{62 (link)–64 (link)} maintained in NeuroCult™ NS-A Proliferation media (#05751, Stemcell). 08-0537 was generously provided by Hai Yan (Duke).
ATRX knockdown was achieved by introducing either a modified FUGW vector (a gift from David Baltimore (Addgene plasmid # 14883)) carrying an shRNA expression cassette against ATRX (shATRX1) (see Supplementary Table 2 for shRNA sequences), a TRIPZ TET-inducible vector (Dharmacon) containing a distinct shRNA against ATRX (shATRX2), or a third shRNA against ATRX (sh590) from the TRC shRNA library (Sigma). shATRX1- and shATRX2-positive cells were FACS-sorted every two passages by fluorescent marker (RFP) for the top 5% of total population to ensure high shRNA expression. Sh590-positive TS 543 cells were subjected to puromycin-based selection.

Wang Y., Yang J., Wild A.T., Wu W.H., Shah R., Danussi C., Riggins G.J., Kannan K., Sulman E.P., Chan T.A, & Huse J.T. (2019). G-quadruplex DNA drives genomic instability and represents a targetable molecular abnormality in ATRX-deficient malignant glioma. Nature Communications, 10, 943.

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Establishing Patient-Derived Glioma Cell Lines

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Three H3.3 K27M mutant patient-derived cell lines - BT245 (8 years old, male), SU-DIPGXIII (6 years old, female), HSJ019 (13 years old, female), and one H3 wild-type cell line - G477 (15 years old, female), were used in this study (Key Resources Table; Table S1). Tumor-derived cell lines were maintained in NeuroCult NS-A proliferation media (StemCell Technologies) supplemented with bFGF (10ng/mL) (StemCell Technologies), rhEGF (20 ng/mL) (StemCell Technologies) and heparin (0.0002%) (StemCell Technologies) on plates coated in poly-L-ornithine (0.01%) (Sigma) and laminin (0.01 mg/mL) (Sigma). All lines tested negative for mycoplasma contamination, checked monthly using the MycoAlert Mycoplasma Detection Kit (Lonza). Tumor-derived cell lines were confirmed to match original samples by STR fingerprinting.

Harutyunyan A.S., Chen H., Lu T., Horth C., Nikbakht H., Krug B., Russo C., Bareke E., Marchione D.M., Coradin M., Garcia B.A., Jabado N, & Majewski J. (2020). H3K27M in Gliomas Causes a One-Step Decrease in H3K27 Methylation and Reduced Spreading within the Constraints of H3K36 Methylation. Cell reports, 33(7), 108390.

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Establishing Patient-Derived Glioma Cell Lines

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