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Epicult b medium

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EpiCult-B medium is a specialized culture medium formulated to support the expansion and maintenance of human and mouse mammary epithelial cells in vitro. The medium is optimized to promote the growth and proliferation of these cell types.

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Lab products found in correlation

Hiseq 4000 sequencing, by Illumina (2 mentions) Fluidigm c1 suspension reagents, by Standard BioTools (2 mentions) Bz x710 microscope, by Keyence (2 mentions) Ultra low attachment 96 well plate, by Corning (2 mentions) Collagenase hyaluronidase, by STEMCELL (2 mentions) Human recombinant bfgf, by Thermo Fisher Scientific (2 mentions) Fibroblasts medium, by ScienCell (2 mentions) Growth factor reduced matrigel matrix, by Corning (2 mentions) Epicult b medium, by Corning (2 mentions) Human recombinant ngf, by Thermo Fisher Scientific (2 mentions) Human recombinant mmp3, by Thermo Fisher Scientific (2 mentions) Recombinant human egf, by Thermo Fisher Scientific (2 mentions) Mammary epithelial growth medium, by Lonza (2 mentions) Epidermal growth factor (egf), by Merck Group (2 mentions) Heparin, by Merck Group (1 mentions) Basic fibroblast growth factor (bfgf), by BD (1 mentions) L calc v1, by STEMCELL (1 mentions) Rock inhibitor y 27632, by Bio-Techne (1 mentions) Dnase 1, by STEMCELL (1 mentions) Geltrex coated, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Epicult-B mouse medium (7 citations) EpiCult-B (4 citations) EpiCult-B Mouse Medium Kit (4 citations) Epicult-B Basal medium (4 citations) EpiCult-B Basal Medium (Human) (2 citations)

16 protocols using epicult b medium

1

Single-cell RNA-seq of Mammary Cell Populations

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For scRNA-seq using the 10X Chromium system, FACS-sorted mammary basal and luminal cell populations were combined. Library generation and Illumina HiSeq-4000 sequencing were performed as previously described (Haensel et al., 2020 (link)). For scRNA-seq using the Fluidigm C1 platform, FACS-sorted basal MECs (Lin−CD49fhighEpCAM+) were washed and resuspended with Epicult-B medium (Stem Cell Technologies; Ca. No. 05610) at a concentration of ~500 cells/μL. Cell suspensions were mixed with Fluidigm C1 Suspension Reagents (Fluidigm 100–5315) at a ratio of 8:2 before loading onto the C1 chip (Fluidigm 100–5760). Bright-field images of captured cells were collected using a Keyence BZ-X710 microscope (Keyence Corporation, Itasca, Illinois, USA). Single-cell RNA isolation and amplification were performed using the Fluidigm C1 Single Cell Auto Prep IFC following the Fluidigm Protocol #100–7168 I1. RNA spike-in controls were omitted. cDNA library preparation was performed following the Fluidigm C1 Protocol #100–7168 I1.
Data analysis was performed using Seurat as we previously described (Haensel et al., 2020 (link)). Heatmaps were based on normalized, but not raw, values of expression to enable direct comparison across runs. A color gradient depicting the expression of each gene in each cell per average for all the cells was generated for each analysis combining biological replicates.
Han Y., Villarreal-Ponce A., Gutierrez G J.r., Nguyen Q., Sun P., Wu T., Sui B., Berx G., Brabletz T., Kessenbrock K., Zeng Y.A., Watanabe K, & Dai X. (2022). Coordinate control of basal epithelial cell fate and stem cell maintenance by core EMT transcription factor Zeb1. Cell reports, 38(2), 110240.
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2

Single-cell RNA-seq of Mammary Cell Populations

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For scRNA-seq using the 10X Chromium system, FACS-sorted mammary basal and luminal cell populations were combined. Library generation and Illumina HiSeq-4000 sequencing were performed as previously described (Haensel et al., 2020 (link)). For scRNA-seq using the Fluidigm C1 platform, FACS-sorted basal MECs (Lin−CD49fhighEpCAM+) were washed and resuspended with Epicult-B medium (Stem Cell Technologies; Ca. No. 05610) at a concentration of ~500 cells/μL. Cell suspensions were mixed with Fluidigm C1 Suspension Reagents (Fluidigm 100–5315) at a ratio of 8:2 before loading onto the C1 chip (Fluidigm 100–5760). Bright-field images of captured cells were collected using a Keyence BZ-X710 microscope (Keyence Corporation, Itasca, Illinois, USA). Single-cell RNA isolation and amplification were performed using the Fluidigm C1 Single Cell Auto Prep IFC following the Fluidigm Protocol #100–7168 I1. RNA spike-in controls were omitted. cDNA library preparation was performed following the Fluidigm C1 Protocol #100–7168 I1.
Data analysis was performed using Seurat as we previously described (Haensel et al., 2020 (link)). Heatmaps were based on normalized, but not raw, values of expression to enable direct comparison across runs. A color gradient depicting the expression of each gene in each cell per average for all the cells was generated for each analysis combining biological replicates.
Han Y., Villarreal-Ponce A., Gutierrez G J.r., Nguyen Q., Sun P., Wu T., Sui B., Berx G., Brabletz T., Kessenbrock K., Zeng Y.A., Watanabe K, & Dai X. (2022). Coordinate control of basal epithelial cell fate and stem cell maintenance by core EMT transcription factor Zeb1. Cell reports, 38(2), 110240.
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3

Mammosphere Assay for Cancer Stem Cell Analysis

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MEC were dissociated into single cells according to standard protocol and cultured in mammosphere media consisting of Epicult-B medium (Stem Cell Technology) containing 5% Matrigel, 10 ng/ml EGF (Sigma), 20 ng/ml bFGF (BD Biosciences), 4 μg/ml Heparin (Sigma), 5% heat inactivated FBS, 5 μM Rock inhibitor Y-27632 (Tocris) (24 (link)). Cells were seeded at 1,000-2,000 per well in 96-well ultralow attachment plates (Corning). The number of mammospheres (solid appearance, >50 um in diameter) was counted 7 days after seeding. Mammospheres were treated in vitro with 10 μg/ml TGFβ neutralizing antibody 1D11, IgG1 isotype control antibody, or 2 μM LY364947. Mammospheres pretreated for one week were dissociated and limiting dilutions were seeded into secondary passages without further treatment to measure self-renewal using L-Calc V1.1.1 (StemCell Technologies).
Martinez-Ruiz H., Illa-Bochaca I., Omene C., Hanniford D., Liu Q., Hernando E, & Barcellos-Hoff M.H. (2016). A TGFβ–miR-182-BRCA1 axis controls the mammary differentiation hierarchy. Science signaling, 9(457), ra118.
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4

Mammosphere Colony Formation Assay

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Single cells obtained by dissociation of primary mammosphere were embedded in Matrigel on 24-well plates and incubated in EpiCult-B medium (Stem Cell Technologies, Vancouver, Canada) containing 1% fetal bovine serum. After 3 weeks, the colony structures generated from Matrigel were counted under a microscope.
Shin D.H., Park J.H., Lee J.Y., Won H.Y., Jang K.S., Min K.W., Jang S.H., Woo J.K., Oh S.H, & Kong G. (2015). Overexpression of Id1 in transgenic mice promotes mammary basal stem cell activity and breast tumorigenesis. Oncotarget, 6(19), 17276-17290.
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5

Isolation and Culture of Triple-Negative Breast Cancer Cells

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Triple-negative human breast cancer cells were isolated and cultured as previously reported (53 (link)). Briefly, tumor tissue was mechanically cut into small pieces (<1 mm3) using scalpels and enzymatically dissociated in a mixture of collagenase/hyaluronidase (STEMCELL Technologies, 07912) for 16 hours at 37°C, followed by further digestion with trypsin (0.25%) for 2 minutes and then 5 units/mL of dispase (STEMCELL Technologies, 07913) and 0.05 mg/mL of DNase I (STEMCELL Technologies, 07900) for 1 minute. After centrifugation at 150g for 5 minutes, cells were seeded at a density of 1 × 105/well onto Geltrex-coated (23 μg of protein per 1 cm2; Thermo Fisher Scientific, A1413202) 6-well plates in human complete EpiCult-B medium (STEMCELL Technologies, 05602, 05630, and 07925). Cells were either further cultured or passaged using trypsin-EDTA (0.25%) for downstream usage.
Wang J., Wang Q., Guan Y., Sun Y., Wang X., Lively K., Wang Y., Luo M., Kim J.A., Murphy E.A., Yao Y., Cai G, & Fan D. (2022). Breast cancer cell–derived microRNA-155 suppresses tumor progression via enhancing immune cell recruitment and antitumor function. The Journal of Clinical Investigation, 132(19), e157248.
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6

Tissue Digestion and Single-cell Isolation

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Tissue samples were minced and digested with collagenase/hyaluronidase (STEMCELL Technologies) in complete EpiCult-B medium supplemented with hydrocortisone (0.48 μg/ml; STEMCELL Technologies) overnight at 37°C. The resulting suspensions were either cryopreserved or further sequentially digested with 0.25% trypsin, dispase (5 mg/ml), and deoxyribonuclease I (1 mg/ml). Single-cell suspensions were collected by filtration through a 40-μm cell strainer.
Karaayvaz-Yildirim M., Silberman R.E., Langenbucher A., Saladi S.V., Ross K.N., Zarcaro E., Desmond A., Yildirim M., Vivekanandan V., Ravichandran H., Mylavagnanam R., Specht M.C., Ramaswamy S., Lawrence M., Amon A, & Ellisen L.W. (2020). Aneuploidy and a deregulated DNA damage response suggest haploinsufficiency in breast tissues of BRCA2 mutation carriers. Science Advances, 6(5), eaay2611.
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7

Mammary Epithelial Cell Organoid Culture

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Matrigel organoid culture was performed as described previously (11 (link)). Briefly, freshly isolated mammary epithelial cells or transduced cells were cultured in complete EpiCult-B medium (STEMCELL Technologies) containing 5% Matrigel (Corning), 5% heat-inactivated FBS, EGF (10 ng/ml), FGF (10 ng/ml), heparin (4 μg/ml), and 5 μM Y-27632. Cells were seeded at 2000 cells per well in 96-well ultralow attachment plates (Corning). Organoids were counted 7 to 14 days after seeding. For FGFR1 inhibition, SU5402 was added to the medium at 10 μM.
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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8

Mammary Cell Isolation and Fractionation

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Mammary cells were prepared as described previously [61 (link)]. Female mice at different ages were euthanized and served as donors of mammary glands. Dissected inguinal and thoracic mammary glands (obtained from mice of indicated age) were digested in dissociation medium (1 part 10× gentle collagenase/hyaluronidase and 9 parts EpiCult-B medium [StemCell Technologies, Vancouver, Canada] supplemented with 5% fetal bovine serum) for 15 h at 37°C in a 5% carbon dioxide incubator. The organoid pellet (enriched with epithelial cells) was treated sequentially in 0.64% ammonium chloride, 0.25% trypsin and ethylenediaminetetraacetic acid, and 5 mg/mL dispase with 0.1 mg/mL DNase I. The cell suspension was filtered through a 40-micron mesh before being labeled with antibodies.
The supernatant (enriched with stromal cells) was either collected for RNAseq analysis or discarded. When collected, it was transferred to a new 50 mL centrifuge tube and centrifuge at 350× g for 5 min.
Dong Q., Gao H., Shi Y., Zhang F., Gu X., Wu A., Wang D., Chen Y., Bandyopadhyay A., Yeh I.T., Daniel B.J., Chen Y., Zou Y., Rebel V.L., Walter C.A., Lu J., Huang C, & Sun L.Z. (2016). Aging is associated with an expansion of CD49fhi mammary stem cells that show a decline in function and increased transformation potential. Aging (Albany NY), 8(11), 2754-2770.
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9

Isolation and Culture of Murine Mammary Epithelial Cells

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Mammary glands from 8 to 12-week-old virgin female mice were enzymatically digested and single cell suspensions of purified mammary epithelial cells (MECs) were obtained following a standard protocol49 (link). Briefly, mammary glands were digested at 37 °C for 1–2 h in Epi-Cult-B medium (Stem Cell Technologies Inc) with 600 U/ml collagenase (Sigma Aldrich) and 200 U/ml hyaluronidase (Sigma Aldrich). After lysis of the red blood cells with NH4Cl, the remaining cells were washed with PBS/0.02% w/v EDTA. Cells were then dissociated with 0.25% w/v trypsin, 0.2% w/v EDTA for 2 min by gentle pipetting, then incubated in 5 mg/ml Dispase II (Sigma Aldrich) plus 1 μg/ml DNase I (Sigma Aldrich) for 5 min, followed by filtration through a 40 μM cell strainer (BD Falcon). MECs were then purified using the EasySep Mouse Mammary Stem Cell Enrichment Kit (Stem Cell Technologies Inc). MECs were seeded on top of 50 or 0.5 kPa Easy Coat hydrogels (Cell guidance system) coated with 10 μg/ml fibronectin and harvested after 24 h.
Bertolio R., Napoletano F., Mano M., Maurer-Stroh S., Fantuz M., Zannini A., Bicciato S., Sorrentino G, & Del Sal G. (2019). Sterol regulatory element binding protein 1 couples mechanical cues and lipid metabolism. Nature Communications, 10, 1326.
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10

Primary Mammary Epithelial Cell Culture

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Fibroblasts/stromal cells were cultured in fibroblasts medium (Science-Cell, 2301) and mammary epithelial cells were cultured in EpiCult-B medium (STEMCELL Technologies, 05610) supplemented with 10 ng ml−1 human recombinant EGF (PeproTech, AF-100-15), 10 ng ml−1 human recombinant bFGF (PeproTech, 100-18B), 5% FBS (vol/vol), and 1% Pen Strep (Hyclone, SV30010; vol/vol). Primary human mammary epithelial cells were seeded in Corning Matrigel Matrix–Growth Factor Reduced (Corning, 354230) and immersed in EpiCult-B medium for coculture studies. For cultures with human recombinant NGF (Peprotech, 450-01) and human recombinant MMP3 (Peprotech, 420-03), 100 ng ml−1 and 0.5 μg ml−1 or 1 μg ml−1 were used in Mammary Epithelial Growth Medium (Lonza, CC-3150), respectively. All cells were grown at 37 °C and at 5% CO2. Antibodies used for FACS Isolation are listed in Supplementary Table 14.
Itoh Y., Esaki T., Kaneshige M., Suzuki H., Cook M., Sokoloff L., Cheng S.Y, & Nunez J. (2001). Brain glucose utilization in mice with a targeted mutation in the thyroid hormone α or β receptor gene. Proceedings of the National Academy of Sciences of the United States of America, 98(17), 9913-9918.
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