B 27 supplement

Manufactured by R&D Systems

B-27 supplement is a serum-free and animal component-free media supplement developed for the growth and maintenance of various cell types, including neurons, neural stem cells, and other neural cell cultures. It is designed to provide a defined and chemically-optimized environment to support cell growth and differentiation.

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Lab products found in correlation

Epidermal growth factor (egf), by R&D Systems (5 mentions) Dmem f12, by Thermo Fisher Scientific (2 mentions) N2 supplement, by Thermo Fisher Scientific (2 mentions) Brain derived neurotrophic factor (bdnf), by R&D Systems (2 mentions) Mtesr1 medium, by STEMCELL (2 mentions) Matrigel, by Corning (2 mentions) Inverted microscope, by Zeiss (2 mentions) N acetylcysteine, by Merck Group (2 mentions) N2 supplement, by R&D Systems (2 mentions) Zen image program, by Zeiss (2 mentions) Noggin, by R&D Systems (2 mentions) Basic fibroblast growth factor (bfgf), by R&D Systems (2 mentions) Pen strep, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Sb202190, by Merck Group (1 mentions) Y 27632, by Merck Group (1 mentions) A83 01, by Merck Group (1 mentions) Jagged 1, by AnaSpec (1 mentions) Laminin, by Thermo Fisher Scientific (1 mentions) Fibroblast growth factor (fgf), by R&D Systems (1 mentions)

7 protocols using b 27 supplement

Induction of Induced Neurons from iPSCs

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HEK293T and SH-SY5Y cells were obtained from ATCC. No additional authentication was performed on these lines. HEK293T cells were maintained in a 37°C incubator with 5% CO₂ in DMEM with Glutamax and HEPES (Thermo Fisher Scientific, cat# 10564–029), 10% fetal bovine serum (vol/vol; Invitrogen, cat# 16000–044), and 1% Pen/Strep (vol/vol; Invitrogen, cat# 15140–122). SH-SY5Y cells were maintained in a 37°C incubator with 5% CO₂ in DMEM/F12 (Thermo Fisher Scientific, cat# 11320033), 10% fetal bovine serum, and 1% Pen/Strep.
We induced neuron differentiation in human iPSC-derived neurons (iNeurons) using a previously established protocol with a Tet-On induction system that allows expression of the transcription factor NGN2 (Bieri et al., 2019 (link)). Briefly, iPS cells were maintained with mTeSR1 medium (Stemcell Technologies, cat# 85850) on Matrigel (Fisher Scientific, cat# CB-40230). On the next day of passage, NGN2 was expressed by adding doxycycline (2μg/mL) and selection with puromycin (2μg/mL) for rapid and highly efficient iNeuron induction. Three days after induction, iNeurons were dissociated and grown in Neurobasal medium containing N-2 supplement (Gibco, cat# 17502048), B-27 supplement, BDNF (R&D Systems) and GDNF (R&D Systems) on Matrigel-coated plates.

Rodriguez C.M., Bechek S.C., Jones G.L., Nakayama L., Akiyama T., Kim G., Solow-Cordero D.E., Strittmatter S.M, & Gitler A.D. (2022). Targeting RTN4/NoGo-Receptor reduces levels of ALS protein ataxin-2. Cell reports, 41(4), 111505.

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Organoid Culture from Intestinal Crypts

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For construction of organoids, 200–500 crypts per well were suspended in Matrigel (Corning) as described.^{58 (link)} Complete ENR medium (all components from Thermo Fisher Scientific unless noted) were comprised of advanced DMEM/F12 (Gibco), antibiotic-antimycotic (×100), 1 mM N-acetyl cysteine (Sigma-Aldrich), B27 supplement, N2 supplement, EGF, Noggin (R&D Systems), R-spondin-1-conditioned medium, and Y-27632 (Sigma). Y-27632 was added to the ENR medium for the first 48–72 h of culture only and then removed during the medium change. The ENR medium was replaced every 2 to 3 days. Colon organoids were cultured in the ENR medium supplemented with Wnt3 conditioned media (WENR). Human colon organoids were cultured in WENR medium supplemented with gastrin, nicotinamide, A83-01, and SB202190 (all from Sigma) as described.^{59 (link)} Isolated ISCs and Paneth cells were co-cultured in ENR medium supplemented with Jagged-1 (1 µM; Anaspec). Wnt-C59 (50 µM; Abcam) was used as a porcupine (PORCN) inhibitor. The surface areas of SI and colon organoids were measured microscopically by taking several random non-overlapping photos of organoids in a well using an inverted microscope (Carl Zeiss). Each photo was analyzed using ImageJ software (NIH) and the Zen image program (Carl Zeiss). Organoid perimeters for area measurements were defined manually using automated ImageJ software.

Kim S., Shin Y.C., Kim T.Y., Kim Y., Lee Y.S., Lee S.H., Kim M.N., O E., Kim K.S, & Kweon M.N. (2021). Mucin degrader Akkermansia muciniphila accelerates intestinal stem cell-mediated epithelial development. Gut Microbes, 13(1), 1892441.

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Culturing Stem Cell Spheroids

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1 × 10⁴ cells were cultured in defined serum-free medium composed of DMEM + F12 medium, 20 ng/ml of EGF (epidermal growth factor; R&D Systems), 20 ng/ml of bFGF (basic fibroblast growth factor; R&D Systems), and B27 supplement (R&D Systems). The cells were seeded in an Ultra-Low Attachment 96 well plate (Corning 3471). Spheroids were resuspended to form secondary and tertiary spheroids. The number of spheroids was counted after 14 days.

Yeo S.Y., Ha S.Y., Lee K.W., Cui Y., Yang Z.T., Xuan Y.H, & Kim S.H. (2017). Twist1 is highly expressed in cancer-associated fibroblasts of esophageal squamous cell carcinoma with a prognostic significance. Oncotarget, 8(39), 65265-65280.

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Otosphere Formation and Hair Cell Differentiation

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To form otosphere, sorted cells as described above were placed at a clonal density (10 cells/μL) onto Ultra-Low Attachment dishes (Corning Life Sciences) and cultured in DMEM/F12 plus N2, B27 supplement, epidermal growth factor (20 ng/mL, R&D Systems), and FGF (10 ng/mL, R&D Systems). After 7–14 days, otospheres were collected and counted.
To analyze cell differentiation, we transferred otospheres onto plates coated with poly-D-ornithine (10 μg/mL, Invitrogen) and laminin (10 μg/mL, Invitrogen), which allow spheres to stick on the bottom. After adhesion, we changed the medium every 2 days, supplemented it with DMEM/F12, N2, and B27 factors, and cultured it for 7–10 days. For the hair cell differentiation generated from LGR5-GFP⁺ supporting cells, it was noted that, after 7–10 days in culture, the green fluorescence of LGR5-GFP⁺ supporting cells was lost, similar to studies by others (Chai et al., 2012 (link), Shi et al., 2012 (link)).

Chen Q., Quan Y., Wang N., Xie C., Ji Z., He H., Chai R., Li H., Yin S., Chin Y.E., Wei X, & Gao W.Q. (2017). Inactivation of STAT3 Signaling Impairs Hair Cell Differentiation in the Developing Mouse Cochlea. Stem Cell Reports, 9(1), 231-246.

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Spheroid Formation from Neural Stem Cells

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1 × 10³ cells were cultured in defined serum-free medium composed of DMEM + F12 medium, 20 ng/ml of EGF (epidermal growth factor; R&D Systems), 20 ng/ml of bFGF (basic fibroblast growth factor; R&D Systems), and B27supplement (R&D Systems). The cells were seeded in an Ultra-Low Attachment 96 well plate (Corning 3471). Spheroids were resuspended to form secondary and tertiary spheroids. The number of spheroids was counted after 14 days.

Yeo S.Y., Ha S.Y., Yu E.J., Lee K.W., Kim J.H, & Kim S.H. (2014). ZNF282 (Zinc finger protein 282), a novel E2F1 co-activator, promotes esophageal squamous cell carcinoma. Oncotarget, 5(23), 12260-12272.

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Isolation and Culture of Intestinal Organoids

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6–8 week-old BALB/C mice were euthanized by decapitation. 10 cm of terminal ileum was taken immediately and washed with pre-cooled (4°C) PBS. Adipose tissue and mesentery in the intestine were removed. Intestinal contents were washed away with 4°C pre-chilled PBS. After the ileum was cut into 2 mm pieces, pipetting was repeated 6 times with PBS. The supernatant was removed after the tissue was precipitated. EDTA-containing PBS was added to precipitates and incubated for 30 min at 4°C on a shaker. After incubation, pipetting was repeated 6 times with PBS until the crypts are dispersed. 200–500 crypts per well were suspended in Matrigel (Corning). Complete ENR medium (all components from Thermo Fisher Scientific) consisted of advanced DMEM/F12 (Gibco), antibiotic and antifungal (×100), 1 mM N-acetylcysteine (Sigma-Aldrich), B27 Supplement, N2 Supplement, EGF, Noggin (R&D Systems), R-spondin-1-conditioned medium was added for the culture. The ENR medium was replaced every 2 to 3 days. The surface area of intestinal organoids was measured from non-overlapping photographs of the organoids randomly taken in a well using an inverted microscope (Carl Zeiss). Each photo was analyzed using ImageJ software (NIH) and the Zen image program (Carl Zeiss)^{50 (link)}.

He K.Y., Lei X.Y., Wu D.H., Zhang L., Li J.Q., Li Q.T., Yin W.T., Zhao Z.L., Liu H., Xiang X.Y., Zhu L.J., Cui C.Y., Wang K.K., Wang J.H., Lv L., Sun Q.H., Liu G.L., Xu Z.X, & Jian Y.P. (2023). Akkermansia muciniphila protects the intestine from irradiation-induced injury by secretion of propionic acid. Gut Microbes, 15(2), 2293312.

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Induction and Manipulation of Human iPSC-Derived Neurons

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We induced neuron differentiation in human iPSC-derived neurons (iNeurons) using a previously established protocol with a Tet-On induction system that allows expression of the transcription factor NGN2 (Bieri et al., 2019) . Briefly, iPS cells were maintained with mTeSR1 medium (Stemcell Technologies, cat# 85850) on Matrigel (Fisher Scientific, cat# CB-40230). On the next day of passage, NGN2 was expressed by adding doxycycline (2µg/ml) and selection with puromycin (2µg/ml) for rapid and highly efficient iNeuron induction. Three days after induction, iNeurons were dissociated and grown in Neurobasal medium containing N-2 supplement (Gibco, cat# 17502048), B-27 supplement, BDNF (R&D Systems) and GDNF (R&D Systems) on Matrigel-coated plates.
At 7 days post neural induction, iNeurons were transduced with virus as above (Mission® shRNA, human RTN4R: TRCN0000061558). 24hr later, a half media change was performed. iNeurons were maintained for 3 days before being re-transduced with a half dose of virus, then maintained for another 5 days prior to harvesting for experimentation. For NEP1-40 treatment, 1mg of NEP1-40 was diluted as above. The peptide was added at 7 days post induction to the final concentration specified along with vehicle (0µM). Cells were maintained for 48hr prior to lysis.

Rodriguez C.M., Bechek S., Jones G.L., Nakayama L., Akiyama T., Kım G., Solow-Cordero D.E., Strittmatter S.M., & Gitler A.D. (2021). Targeting RTN4/NoGo-Receptor reduces levels of ALS protein ataxin-2.

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