B27 without vitamin a

Manufactured by Thermo Fisher Scientific

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B27 without vitamin A is a cell culture supplement manufactured by Thermo Fisher Scientific. It is designed to support the growth and maintenance of neuronal and other cell types in vitro. The supplement provides a defined set of nutrients, growth factors, and other components necessary for cell culture, but without the inclusion of vitamin A.

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Vitamin A (15 citations) B27-Vitamin A (10 citations)

92 protocols using b27 without vitamin a

Induced Pluripotent Stem Cell Differentiation

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iPSC medium: KO-DMEM (Life Technologies, Carlsbad, CA, USA), supplemented with 20% KnockOut Serum Replacement (Life Technologies), 1X MEM non-essential amino acids (Life Technologies), 2 mM Glutamine (Life Technologies), 50 µM β-mercaptoethanol (Life Technologies), and 10 UI/mL gentamycin (Life Technologies).
Differentiated medium: DMEM/F12 (Life Technologies), 2% B27 without vitamin A (Life Technologies), 5 µg/mL heparin (Sigma-Aldrich, Saint-Quentin Fallavier, France), and 100 µM β-mercaptoethanol (Life Technologies).
Neural induction medium: 1:1 DMEM/F12 (Life Technologies) and Neurobasal A (Life Technologies), 1% N2 supplement (Life Technologies), 2% B27 without vitamin A (Life Technologies), and 100 µM β-mercaptoethanol (Life Technologies).

Faye P.A., Vedrenne N., Miressi F., Rassat M., Romanenko S., Richard L., Bourthoumieu S., Funalot B., Sturtz F., Favreau F, & Lia A.S. (2020). Optimized Protocol to Generate Spinal Motor Neuron Cells from Induced Pluripotent Stem Cells from Charcot Marie Tooth Patients. Brain Sciences, 10(7), 407.

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Culturing Primary Glioblastoma Cell Lines

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GBM cancer stem cell line 0308 was provided by Howard Fine (Department of Neurology, Weill Cornell Medicine, New York, New York, USA) (37 (link), 77 (link)). TS667 from a patient with primary GBM was derived in-house (78 (link)), as was MGG8 from a patient with primary GBM (79 (link)). 0308 and TS667 cells were cultured in Neurobasal media (Life Technologies) supplemented with 0.5× B27 without vitamin A (Thermo Fisher), 0.5× N2 supplement (Thermo Fischer), 2 mM L-glutamine (Thermo Fisher), 1 mM sodium pyruvate (Thermo Fisher), 50 μg/ml EGF (Peprotec), and 50 μg/ml basic FGF (Peprotec). MGG8 cells were cultured in Neurobasal media (Life Technologies) supplemented with 1× B27 without vitamin A, 1× N2 supplement, 3 mM GlutaMAX (Gibco), 5 mg/ml heparin (Stem Cell Technologies), 20 ng/ml EGF (Peprotec), and 20 μg/ml basic FGF (Peprotec).

Galdieri L., Jash A., Malkova O., Mao D.D., DeSouza P., Chu Y.E., Salter A., Campian J.L., Naegle K.M., Brennan C.W., Wakimoto H., Oh S.T., Kim A.H, & Chheda M.G. (2021). Defining phenotypic and functional heterogeneity of glioblastoma stem cells by mass cytometry. JCI Insight, 6(4), e128456.

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Neural Induction of Stem Cells

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Neural induction was initiated three days after integration into silk foam using a modified version of a dual-SMAD inhibition protoc. 28, 29 The cells were cultured in neural induction medium (NIM) consisting of 50% Neurobasal medium (ThermoFisher) and 50% DMEM/F12 (ThermoFisher) supplemented with 0.5× B-27 without vitamin A (ThermoFisher), 0.5× N2 (ThermoFisher), 100 µM 2-mercaptoethanol (Sigma) and GlutaMAX (Life Technologies) with the addition of recombinant human Noggin, 500 ng ml -1 (R&D) and SB431542, 10 µM (Tocris) with daily medium change. After seven days, NIM was replaced by neuronal progenitor medium (NPDM); 50% Neurobasal medium (ThermoFisher Scientific) and 50% DMEM/F12 (ThermoFisher) supplemented with 0.5× B-27 without vitamin A (ThermoFisher), 0.5× N2 (ThermoFisher), 100 µM 2-mercaptoethanol (Life Technologies) and GlutaMAX (Life Technologies). Medium was changed every second or third day for the remaining culture time.

Åstrand C ., Chotteau V ., Falk A , & Hedhammar M . (2020). Assembly of FN-silk with laminin-521 to integrate hPSCs into a three-dimensional culture for neural differentiation. Biomaterials science, 8(9).

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Culturing Prostaspheres from LNCaP-abl Cells

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LNCaP-abl cells were resuspended, dissociated into single cells and were cultured in 6-well ultra-low attachment culture plates (Corning, Amsterdam, The Netherlands) with DMEM/F-12 (Gibco, Carlsbad, CA) supplemented with 1% L-Glutamax, 1% P/S, 20 ng/μL human epidermal growth factor (hEGF; Gibco), 20 ng/μL basic fibroblasts growth factor (bFGF; Gibco), 1× B27 without vitamin A (Invitrogen), heparin sodium, 1× insulin-transferrin-selenium A (Invitrogen), 1× non-essential amino acids (Invitrogen), 0.1 mM β-mercaptoethanol (Sigma) and 10³ U/mL leukemia inhibitory factor (LIF; Millipore, Billerica, MA). Besides these, the concentration of E2 was 1 nM in the control group and 10 nM in the E2 group. Sphere cultures were seeded at a density of 2 × 10³ cells/mL and culture media was fully replaced every 96 h. Parental and prostasphere cultures were propagated for 12 days before the spheres were enzymatically dissociated using StemPro Accutase (Gibco) for second and third generation cell culture for 7 days, separately. Spheres were imaged and visualized via Olympus CX41 microscopy to evaluate the volume, or were collected for qRT-PCR or immunofluorescence detection.

Shen Y., Cao J., Liang Z., Lin Q., Wang J., Yang X., Zhang R., Zong J., Du X., Peng Y., Zhang J, & Shi J. (2019). Estrogen receptor α-NOTCH1 axis enhances basal stem-like cells and epithelial-mesenchymal transition phenotypes in prostate cancer. Cell Communication and Signaling : CCS, 17, 50.

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Glioblastoma Clonal Cell Culture Protocol

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The GBM clonal cell culture, U3065−c271 [28 (link)], was cultured in neural stem cell media (1:1 mix of DMEM-F12 GlutaMAX medium and Neurobasal medium (Life Technologies/GIBCO-Invitrogen) containing 1% penicillin G/streptomycin sulfate (Sigma-Aldrich, St. Louis, MO), supplemented with B-27 without vitamin A (1:50; Invitrogen), N2 supplement (1:100; Invitrogen), 10 ng/mL EGF and 10 ng/mL FGF-2 (PeproTech, Rocky Hill, NJ). Cells were seeded in poly-L-ornithine (P4957, Sigma-Aldrich) and laminin (L2020, Sigma-Aldrich) coated 384-well plates (164688, Thermo Fisher Scientific) at a density of 1000 cells/well using a BioMek 4000 (Beckman Coulter). All cells were seeded 24 h prior to treatment with compounds.

Chantzi E., Jarvius M., Niklasson M., Segerman A, & Gustafsson M.G. (2019). COMBImage2: a parallel computational framework for higher-order drug combination analysis that includes automated plate design, matched filter based object counting and temporal data mining. BMC Bioinformatics, 20, 304.

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Cerebral Organoid Differentiation from hESCs

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Organoid-like cerebral structures were differentiated from hESCs as described previously (Lancaster et al., 2013 (link); Lancaster and Knoblich, 2014 (link)). Briefly, hESCs cultured on MEF feeders were digested into single cells. EBs were formed from 2 × 10⁴ cells in a U-bottom ultralow attachment 96-well plate (Corning) in low FGF-2 hESC medium supplemented with 50 μmol/L Y-27632. After four days, FGF-2 and Y-27632 were deprived for another two days. Then, the EBs were transferred to ultralow attachment 24-well plates (Corning) in neural induction medium (DMEM/F12 supplemented with 1% N2, 2 mmol/L GlutaMAX, 0.1 mmol/L NEAA, 1% penicillin/streptomycin and 1 μg/mL heparin (Selleck)) for four days. The EBs were packaged into Matrigel droplets and further cultured in 6-well plates in cerebral organoid differentiation medium (50% DMEM/F12 and 50% Neurobasal, with additional 0.5% N2, 1% B27 without vitamin A (Invitrogen), 2 mmol/L GlutaMAX, 0.05 mmol/L NEAA, 1% penicillin/streptomycin, 2.75 μg/mL insulin (Sigma) and 50 μmol/L β-mercaptoethanol (Sigma)) for four days. Then, the plates were placed on an orbital shaker (shaking at 85 rpm) installed in the incubator, and B27 without vitamin A was replaced by normal B27. After approximately 40 days, the structures were fixed by 4% (w/v) paraformaldehyde for cryosectioning and immunostaining or collected directly for RNA extraction.

Zhang X., Liu Z., Liu X., Wang S., Zhang Y., He X., Sun S., Ma S., Shyh-Chang N., Liu F., Wang Q., Wang X., Liu L., Zhang W., Song M., Liu G.H, & Qu J. (2019). Telomere-dependent and telomere-independent roles of RAP1 in regulating human stem cell homeostasis. Protein & Cell, 10(9), 649-667.

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Mouse Tongue-Derived Organoid Culture

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For mouse-derived organoids, tongue tissue was obtained from C57BL/6 mice. Excess fat and muscle tissue were removed to enrich for epithelial cells. The tissue was then finely minced into 2–3 mm pieces on ice, washed twice in DMEM/F-12 (Invitrogen) containing 1X Primocin (InvivoGen), followed by ACK solution (Thermo Fisher Scientific) to remove red blood cell contamination. Minced tissues were washed with DMEM/F-12 media and resuspended with Matrigel (cold Cultrex growth factor reduced Basement Membrane Extract (BME) type 2, Trevigen). Droplets of approximately 40 μL were plated on the bottom of preheated 24-well culture plates (E&K Scientific). Plates were incubated at 37°C for 30 min to allow BME to solidify. Organoid culture media (EN media) containing DMEM/F-12 supplemented with 10% Noggin-conditioned media, nicotinamide (10 mM, Sigma), N-acetylcysteine (1 mM, Sigma), B-27 without vitamin A (1X, Invitrogen), Pen-Strep (1X, Invitrogen) and EGF (50 ng/mL, R&D Systems) was added. The medium was changed every 2–3 days and organoids were split once every 2–4 weeks.

Parikh A., Shin J., Faquin W., Lin D.T., Tirosh I., Sunwoo J.B, & Puram S.V. (2020). Malignant cell-specific CXCL14 promotes tumor lymphocyte infiltration in oral cavity squamous cell carcinoma. Journal for Immunotherapy of Cancer, 8(2), e001048.

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Metabolomics of Oligodendrocyte Differentiation

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All the endogenous metabolites used in this study were from Sigma-Aldrich. Benztropine, miconazole and triiodothyronine (T3) were purchased from Sigma-Aldrich (Saint Louis, MO). All the chemicals used in this study have a purity ≥ 95%.
Rat primary optic nerve OPCs were isolated by panning (>99% A2B5⁺) and cultured in poly-d-lysine (10 µg/ml)-coated TC dishes in OPC culture media (Neurobasal Media, Invitrogen) supplemented with B27 without vitamin A (Invitrogen), 1× nonessential amino acids, 1× Glutamax, 1× anti-anti, β-mercaptoethanol and PDGFαα (50 ng/ml; Peprotech) at 37 °C with 5% CO₂. The culture medium was replaced every 48 h, and cells were collected before the confluency reached 60% to maintain a naive state. For differentiation, OPCs were plated at 1 million cells in one 10-cm Petri dish filled with differentiation media, which is identical to the culture media but with PDGF-AA at 2 ng/ml. T3 and DMSO were used as the positive control and negative control, respectively. Five replicates of cells were collected at different times (0, 3 and 6 d) for metabolomics studies.

Beyer B.A., Fang M., Sadrian B., Montenegro-Burke J.R., Plaisted W.C., Kok B.P., Saez E., Kondo T., Siuzdak G, & Lairson L.L. (2017). Metabolomics-based discovery of a metabolite that enhances oligodendrocyte maturation. Nature chemical biology, 14(1), 22-28.

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Mammosphere Culture and Differentiation

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In the present study we also adopted the SFD assay developed for mouse MaSC identification32 (link) for stem/progenitor cell characterization in marmosets and baboons. The detailed method has been described previously32 (link). In brief, sorted cells were cultured in ultralow attachment 96-well plates (Corning) with human MammoCult complete medium (StemCell Technologies) supplemented with 2% B27 without vitamin A (Invitrogen), 20 ng/mL bFGF, 20 ng/mL EGF, 10 μg/mL heparin, 10 μg/mL insulin, 1 μg/mL hydrocortisone, 50 μg/mL gentamycin (referred to as mammosphere or MMS medium) at 37 °C in a 5% CO2 incubator. Cells were plated at two densities of 10,000 and 20,000 cells/well. After 7–10 days of suspension culture, spheres were counted and about 50 spheres were handpicked under a dissecting microscope and resuspended in 60 μl chilled Matrigel (BD Biosciences) for sphere differentiation. The sphere-Matrigel drop was allowed to solidify inside a 37 °C incubator for 15 min, covered with MMS medium supplemented with 5% FBS, and incubated at 37 °C for 9 days before examination.

Wu A., Dong Q., Gao H., Shi Y., Chen Y., Zhang F., Bandyopadhyay A., Wang D., Gorena K.M., Huang C., Tardif S., Nathanielsz P.W, & Sun L.Z. (2016). Characterization of mammary epithelial stem/progenitor cells and their changes with aging in common marmosets. Scientific Reports, 6, 32190.

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Glioblastoma Tumor Sampling and Culture

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Glioblastoma samples were obtained from surgical resection from patients at Duke University or Case Western Reserve University with informed consent in accordance with the Cleveland Clinic Institutional Review Board-approved protocol 090401. Prior to use, all samples were reviewed and verified by a neuropathologist. All patient studies were conducted in accordance with the Declaration of Helsinki. GSC23 was acquired via a material transfer agreement from The University of Texas MD Anderson Cancer Center (Houston, TX). GSCs were cultured in Neurobasal media (Invitrogen) supplemented with B27 without vitamin A (Invitrogen), EGF, and bFGF (20 ng/mL each: R&D Systems), sodium pyruvate, and Glutamax. Short tandem repeat analyses were performed to authenticate the identity of each tumor model used in this article both annually and prior to high-throughput sequencing. Cells were stored at −160°C when not being cultured. To minimize cell culture-based artifacts, patient-derived xenografts were produced and propagated as a renewable source of tumor cells for study. Cultured cells underwent PCR testing for mycoplasma routinely every 6 months, and all RNA-sequencing data were evaluated for mycoplasma content during quality control.

Sen A., Prager B.C., Zhong C., Park D., Zhu Z., Gimple R.C., Wu Q., Bernatchez J.A., Beck S., Clark A.E., Siqueira-Neto J.L., Rich J.N, & McVicker G. (2021). Leveraging Allele-Specific Expression for Therapeutic Response Gene Discovery in Glioblastoma. Cancer Research, 82(3), 377-390.

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