Epicult b mouse medium

Manufactured by STEMCELL

Epicult-B mouse medium is a culture medium designed for the maintenance and expansion of mouse embryonic stem cells (mESCs) and induced pluripotent stem cells (iPSCs).

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EpiCult-B medium (16 citations) EpiCult-B (4 citations) EpiCult-B Mouse Medium Kit (4 citations) Mouse EpiCult-B (2 citations)

7 protocols using epicult b mouse medium

Mammary Epithelial Cell GPR40 Agonist Assay

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Mouse mammary epithelial cells were cultured in FBS‐free complete EpiCult‐B medium (Mouse) (#05610; STEMCELL Technologies) as described above and treated with 100 nmol/L GPR40 agonist (TAK‐875; ChemScene) or DMSO (Nacalai Tesque).

Kulathunga N., Kohno S., Linn P., Nishimoto Y., Horike S., Zaraiskii M.I., Kumar S., Muranaka H, & Takahashi C. (2020). Peripubertal high‐fat diet promotes c‐Myc stabilization in mammary gland epithelium. Cancer Science, 111(7), 2336-2348.

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Mouse Mammary Cells Lipid Treatment

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Mouse mammary epithelial cells were cultured in FBS‐free complete EpiCult‐B medium (Mouse) (#05610; STEMCELL Technologies) as described above and treated with 3 mmol/L BSA‐conjugated OA (stock solution; #O3008) (Sigma‐Aldrich) or 30% w/v fatty acid‐free BSA (015‐23871; Wako) alone for 48 hours. For the PA treatment, BSA‐conjugated PA 3 mmol/L stock solution was prepared according to a protocol described previously using sodium palmitate (P9767; Sigma‐Aldrich) and BSA (A7030; Sigma‐Aldrich) and treated for 48 hours.²⁶

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Mammosphere Formation and Transplantation

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Following primary mammary epithelial cell extraction and infection, cells were added to mammosphere medium plated in suspension for 3 d. The mammosphere medium consisted of Epicult-B mouse medium (Stem Cell Technologies, 05610), knockout serum replacement (Gibco, 10828010), penicillin/streptomycin, 20 ng/mL EGF, 20 ng/mL FGF, and 4 μg/mL heperan sulfate. Next, the cells were spun down, resuspended in PBS with 10% GelTrex and 10% Trypan blue, and injected into the cleared mammary fat pad of a 3-wk-old FVB female mouse. Each injection was 10–12 μL in volume.

Halaoui R., Rejon C., Chatterjee S.J., Szymborski J., Meterissian S., Muller W.J., Omeroglu A, & McCaffrey L. (2017). Progressive polarity loss and luminal collapse disrupt tissue organization in carcinoma. Genes & Development, 31(15), 1573-1587.

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Mammary Epithelial Organoid Assay

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Epithelial cells were harvested from the mammary glands of 8 to 12-wk-old MIC mice and grown as organoids as described previously (28 (link)). Roughly 10,000 mammary epithelial cells were cultured in eight-well chambers in the organotypic medium consisted of Epicult-B mouse medium (Stem Cell Technologies, 05610), knockout serum replacement (Gibco, 10828010), penicillin/streptomycin, 10 ng/mL EGF, 25 μg/mL insulin (Sigma, 10156), and 1 μg/mL hydrocorticone for 6 d to allow formation of acinar structures. Subsequently, doxycycline was added to growth media either in the presence or absence of drug treatments for 8 d, with media changes every 48 h. The following inhibitors, along with their working doses, were used: GSK-126 (MedChem Express, Cat#HY-13470, 2 μM), EPZ6438 (MedChem Express, Cat#13803, 2 μM), Torin-1 (Selleckchem, Cat#S2827, 250 nM), Rapamycin (MedChem Express, Cat#HY-10219, 100 nM), IWP-2 (STEMCELL Technologies, Cat#721220, 20 nM), MS-177 (MedChem Express, Cat#HY-148333, 5 μM), A-395 (MedChem Express, Cat#HY-101512, 2.5 μM), 3-Deazaneplanocin A hydrochloride (DZNep) (MedChem Express, Cat#HY-10442, 2 μM), and Pictilisib (GDC-0941) (MedChem Express, Cat#HY-50094, 5 μM).

Liu L., Xiao B., Hirukawa A., Smith H.W., Zuo D., Sanguin-Gendreau V., McCaffrey L., Nam A.J, & Muller W.J. (2023). Ezh2 promotes mammary tumor initiation through epigenetic regulation of the Wnt and mTORC1 signaling pathways. Proceedings of the National Academy of Sciences of the United States of America, 120(33), e2303010120.

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Murine 2D Colony-Forming Assay

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All murine 2D colony-forming assays were performed using female FVB wild-type mice. In brief, total dissociated mammary cells or FACS-purified basal and luminal cells were seeded together with irradiated NIH 3T3 cells. Cells were allowed to adhere, and either vehicle control (0.1% DMSO) or the indicated concentrations of epigenomic inhibitor were added. Cells were cultured for 7 d at 5% oxygen to allow basal colony growth in either Epicult-B mouse medium (Stem Cell Technologies) supplemented with 5% FBS, 10 ng/ml EGF, 20 ng/ml basic FGF, 4 µg/ml heparin, and 5 µM ROCK inhibitor (Millipore) or DMEM/F12 (1:1) supplemented with 10% FBS, 5 µg/ml insulin (Thermo Fisher Scientific), 10 ng/ml EGF, 10 ng/ml cholera toxin, 1.8 × 10⁴ M adenine (Sigma), 0.5 µg/ml hydrocortisone, and 10 µM ROCK inhibitor (Millipore). Growth factors and hydrocortisone were obtained from Stem Cell Technologies.

Casey A.E., Sinha A., Singhania R., Livingstone J., Waterhouse P., Tharmapalan P., Cruickshank J., Shehata M., Drysdale E., Fang H., Kim H., Isserlin R., Bailey S., Medina T., Deblois G., Shiah Y.J., Barsyte-Lovejoy D., Hofer S., Bader G., Lupien M., Arrowsmith C., Knapp S., De Carvalho D., Berman H., Boutros P.C., Kislinger T, & Khokha R. (2018). Mammary molecular portraits reveal lineage-specific features and progenitor cell vulnerabilities. The Journal of Cell Biology, 217(8), 2951-2974.

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Tumor Cell Isolation Protocol

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Single cell suspensions were prepared from tumors by mincing into 1mm³ fragments and dissociating in Epicult-B Mouse Medium (Stem Cell Technologies, #05610) supplemented with 5% fetal bovine serum (FBS), 10 ng/mL epidermal growth factor (EGF), 300 U/mL collagenase, and 100 U/mL hyaluronidase (Stem Cell Technologies, #07912) at 37°C for one hour. Red blood cells were lysed using ammonium chloride, and then cells were incubated for one minute in trypsin-EDTA (Stem Cell Technologies, #07901) containing 5 mg/mL dispase (Stem Cell Technologies, #07913) and 0.1 mg/mL DNase I (Stem Cell Technologies, #07900), and filtered through 40 μm strainers.

Shea M.P., O’Leary K.A., Fakhraldeen S.A., Goffin V., Friedl A., Wisinski K.B., Alexander C.M, & Schuler L.A. (2018). Anti-estrogen therapy increases plasticity and cancer stemness of prolactin-induced ERα+ mammary carcinomas. Cancer research, 78(7), 1672-1684.

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Culturing Primary Mammary Epithelial Cells

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Primary mammary epithelial cells were cultured in EpiCult-B mouse medium (STEMCELL Technologies) supplemented with proliferation supplements, recombinant human epidermal growth factor (EGF; 10 ng/ml), recombinant human basic fibroblast growth factor (10 ng/ml), heparin (4 μg/ml), and penicillin/streptomycin. HMLE cells were cultured in a 1:1 mixture of DMEM/F-12 supplemented with 10% FBS, insulin (0.01 mg/ml), hydrocortisone (0.48 μg/ml), and complete mammary epithelial cell growth medium (MEGM) supplemented with bovine pituitary hormone (Lonza). For activating tetracycline-inducible Snail and Zeb1 expression, cells were grown with doxycycline hyclate (1 μg/ml; Sigma-Aldrich) in medium. For all ciliogenesis assays, cells were grown until high confluence and DMEM/F12 was used to serum starve the cells. For FGFR1 inhibition, cells were treated with SU5402 (Sigma-Aldrich) in DMEM/F12 medium.

Wilson M.M., Callens C., Le Gallo M., Mironov S., Ding Q., Salamagnon A., Chavarria T.E., Viel R., Peasah A.D., Bhutkar A., Martin S., Godey F., Tas P., Kang H.S., Juin P.P., Jetten A.M., Visvader J.E., Weinberg R.A., Attanasio M., Prigent C., Lees J.A, & Guen V.J. (2021). An EMT–primary cilium–GLIS2 signaling axis regulates mammogenesis and claudin-low breast tumorigenesis. Science Advances, 7(44), eabf6063.

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