Complete mammocult medium

Manufactured by STEMCELL

Sourced in United States, Canada

Complete MammoCult Medium is a fully supplemented culture medium designed to support the growth and maintenance of primary human mammary epithelial cells.

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Lab products found in correlation

Six well ultralow attachment plates, by Corning (4 mentions) Axio observer a1, by Zeiss (3 mentions) Withhistochoice mb tissue fixative, by Avantor (2 mentions) Immuno bed resin, by Polysciences (2 mentions) Ultra low attachment plate, by Corning (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) Ultra low attachment 96 well plate, by Corning (2 mentions) Ultra low attachment 6 well plate, by Corning (2 mentions) Tariguidar, by Selleck Chemicals (2 mentions) Ultra low attachment six well plate, by Corning (2 mentions) Methylcellulose, by STEMCELL (2 mentions) Mammocult basal medium, by STEMCELL (2 mentions) Heparin, by STEMCELL (2 mentions) Hydrocortisone, by STEMCELL (2 mentions) Phase contrast microscope, by Olympus (2 mentions) Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions) Doxorubicin dox, by Selleck Chemicals (1 mentions) Slide a lyzer dialysis cassette, by Thermo Fisher Scientific (1 mentions) Propidium iodide, by Revvity (1 mentions) Leptin, by R&D Systems (1 mentions)

Spelling variants of this product

MammoCult medium (95 citations) MammoCult (24 citations) Complete MammoCult™ Human Medium (3 citations)

31 protocols using complete mammocult medium

Mammosphere Formation Assay with APR-246

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MDA-MB-435 and MDA-MB-231 cells were grown in 100 mm dishes in DMEM/F12 medium supplemented with 10% FBS to 60% confluence. Cells were then washed twice in PBS, harvested and counted. Cells (5×10³) in 0.1 mL complete Mammocult™ medium (STEMCELL Technologies) were seeded onto ultra-low adherent six-well plates (STEMCELL Technologies) containing 1.9 mL/well of complete Mammocult medium supplemented with APR-246 at the final concentrations indicated. For controls, an equal volume of PBS (vehicle) was added to the medium. Incubations were carried out in triplicate. Every 48 hrs, cells were retreated with additional 1 mL of fresh APR-246 (or vehicle) in complete Mammocult medium. Images of mammospheres were captured by EVOS light microscopy (10x objective) (Waltham, MA, USA) on days 2, 4, and 6 of treatment. Mammospheres≥100 μm were quantified.

Liang Y., Besch-Williford C., Cook M.T., Belenchia A., Brekken R.A, & Hyder S.M. (2019). APR-246 alone and in combination with a phosphatidylserine-targeting antibody inhibits lung metastasis of human triple-negative breast cancer cells in nude mice. Breast Cancer : Targets and Therapy, 11, 249-259.

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Rat Model of Pancreatic Cancer Metastasis

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A previously developed rat model of pancreatic cancer was used for this study with all work performed in accordance with BUSM institutional guidelines. ³¹ Briefly, Panc-1 cells were harvested in log phase and subcultured in complete MammoCult medium (Stem Cell Technologies, BC, Canada) in 5% CO₂ in a humidified incubator at 37 °C. After 2–3 weeks in culture, Panc-1 cells were harvested and plated in complete MammoCult medium containing 0.5% methylcellulose (Stem Cell Technologies, BC, Canada) in 100 mm ultralow attachment plates. Peritoneal tumors were developed from septenary CSCs. The Panc-1-CSC-derived peritoneal tumor model was developed in 4–5 week-old nude^nu/nu female rats (Charles River Laboratories, MA). The intraperitoneal tumor implantation was performed in anesthetized animals using isoflurane maintained at 1–1.5%. Intraperitoneal injection of 2 × 10⁶ CSCs suspended in 1 mL of M2 media was performed under sterile conditions.

Colby A.H., Berry S.M., Moran A.M., Pasion K.A., Liu R., Colson Y.L., Ruiz-Opazo N., Grinstaff M.W, & Herrera V.L. (2017). Highly Specific and Sensitive Fluorescent Nanoprobes for Image-Guided Resection of Sub-Millimeter Peritoneal Tumors. ACS nano, 11(2), 1466-1477.

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Culturing and Characterizing Cancer Stem Cells

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Panc1 and U87-cells in log phase were harvested and subcultured in complete MammoCult® medium (Stem Cell Technologies, BC, CANADA) in 5% CO2 humidified incubator at 37°C. After 2–3 weeks in culture, Panc1 and U87 cells were harvested and plated in complete MammoCult® medium containing 0.5% Methylcellulose (Stem Cell Technologies, BC, CANADA) in 100 mm ultra-low attachment plates. In vitro experiments were performed using quinary CSCs; xenograft tumors were developed from septenary CSCs. Functional validation of CSC stocks was done through demonstration of maintenance of self-renewal in suspension cultures and anchorage independence through 7 passages, increased tumorigenicity over non-CSC cells, tumor vasculogenesis and tumor cell heterogeneity in xenograft tumors.

Herrera V.L., Decano J.L., Tan G.A., Moran A.M., Pasion K.A., Matsubara Y, & Ruiz-Opazo N. (2014). DEspR Roles in Tumor Vasculo-Angiogenesis, Invasiveness, CSC-Survival and Anoikis Resistance: A ‘Common Receptor Coordinator’ Paradigm. PLoS ONE, 9(1), e85821.

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Cancer Stem Cell Spheroid Culture

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Tumor self-renewing and anchorage-independent spheroids were obtained by culturing breast cancer cells MCF7, Hs587T and MDA231; melanoma cells SBcl2 and FM6; human breast cancer stem cells and pancreatic cancer stem cells in stem cell-selective conditions according to the manufacturer’s instructions (StemCell Technologies, Tukwila, WA). Briefly, cancer and cancer stem cells were propagated in 6-well ultra-low adherent plates (Corning) in Complete MammoCult Medium (Human) by adding 50 mL of MammoCult Proliferation Supplements to 450 mL of MammoCult Basal Medium (StemCell Technologies). The following were added to obtain Complete MammoCult Medium: 4 ug/mL Heparin (StemCell Technologies), 0.48 μg/mL hydrocortisone (StemCell Technologies), 200 U penicillin/ml; and 200 μg streptomycin/ml (Invitrogen).

Chen Z., Forman L.W., Williams R.M, & Faller D.V. (2014). Protein kinase C-delta inactivation inhibits the proliferation and survival of cancer stem cells in culture and in vivo. BMC Cancer, 14, 90.

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Mammosphere Formation Assay for MCF10A Cells

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MCF10A human breast epithelial cells were suspended in MammoCult complete medium containing growth supplements heparin sulfate and hydrocortisone according to the manufacturer’s instructions (Stem Cell Technologies). Mammospheres were cultured according to the MammoCult product sheet. In brief, the cell suspension was passed through a 45-µm cell strainer to obtain single cells. 10,000 MCF10A cells were seeded into each well of a 6-well ultra-low adherence dish (Stem Cell Technologies) containing 2 ml MammoCult complete medium. After 7 d in culture at 5% CO₂ at 37°C, spheres >60 µm in diameter were counted.

Kulic I., Robertson G., Chang L., Baker J.H., Lockwood W.W., Mok W., Fuller M., Fournier M., Wong N., Chou V., Robinson M.D., Chun H.J., Gilks B., Kempkes B., Thomson T.A., Hirst M., Minchinton A.I., Lam W.L., Jones S., Marra M, & Karsan A. (2015). Loss of the Notch effector RBPJ promotes tumorigenesis. The Journal of Experimental Medicine, 212(1), 37-52.

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Leptin and IONP-LPrA2 Modulate Tumorsphere Formation

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MDA-MB-231 cells were seeded at 5 × 10³-2 × 10⁴ cells/mL in low attachment plates and grown for 1-2 wk in Mammocult complete medium (Stem Cell Technologies) supplemented with heparin and hydrocortisone and treated with leptin (1.2 nmol/L), or IONP-LPrA2 (0.0036 pmol/L) plus leptin (1.2 nmol/L). Basal tumorspheres served as untreated controls. The tumorspheres were visually assessed by light microscopy. The size of the tumorspheres were determined and the number of tumorspheres were counted manually in triplicate.

Harmon T., Harbuzariu A., Lanier V., Lipsey C.C., Kirlin W., Yang L, & Gonzalez-Perez R.R. (2017). Nanoparticle-linked antagonist for leptin signaling inhibition in breast cancer. World Journal of Clinical Oncology, 8(1), 54-66.

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Nanoparticle-Based Breast Cancer Treatment

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IONPs were obtained from Ocean Nanotech San Diego, CA. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC), Sulfo-NHS, and other chemicals were purchased from Sigma Aldrich St. Louis, MO. Ob-R (sc-8325), Cyclin D1 (sc-246), pSTAT3 (sc-8059), STAT3 (sc-8019) antibodies were purchased from Santa Cruz Biotechnology Santa Cruz, CA. Anti-rabbit and anti-mouse conjugated to horseradish peroxidase were obtained from Bio-Rad Laboratories Hercules, CA. Dulbecco’s Modified Eagles Medium (DMEM), Iscove’s Modified Dulbecco’s Medium (IMEM), Protease and Phosphatase Inhibitor cocktails, Penicillin/Streptomycin, Slide-a-lyzer dialysis cassette, and Western blotting chemiluminescence substrate were purchased from Thermo Fisher Scientific Rockford, IL. Mammocult complete medium was obtained from Stem Cell Technologies Vancouver, BC. Fetal bovine serum was obtained from Med Supply Partners Atlanta, GA. Leptin was purchased from R and D Systems Minneapolis, MN. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) kit was purchased from Molecular Probes Eugene, OR. Annexin V/fluorescein Isothiocyanate (FITC) and propidium iodide (PI) were obtained from Nexcelom Bioscience Boston, MA. Cisplatin (Cis) was purchased from Millipore Billerica, MA. Cyclophosphamide (CTX), paclitaxel (PTX), and doxorubicin (Dox) were obtained from SelleckChem Houston, TX.

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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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Ovarian Cancer Cell Spheroid Generation

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OC cell lines or primary cells were seeded at a concentration of 25,000
cells/ml in Mammocult complete medium (StemCell Technologies) and ultra-low
attachment plates (Corning, Corning, NY, USA). Spheroids were trypsinized every
7 days and re-plated to generate successive generations. To observe spheroid
morphology, A2780, SKOV3, IGROV1, and primary cells were cultured in a rotating
bioreactor (Synthecon, Houston, TX, USA) until 200-400 μM compact
spheroids were visible (10-30 days). They were harvested, preserved with
Histochoice MB Tissue Fixative (Amresco, Solon, OH, USA) for 1 hour, and
embedded in Immuno-bed resin (Polysciences, Warrington, PA, USA). Two micron
thick sections were cut on an ultramicrotome and stained with 0.1% methylene
blue/0.15% basic fuchsin in 50% methanol.

Condello S., Morgan C.A., Nagdas S., Cao L., Turek J., Hurley T.D, & Matei D. (2014). Beta-Catenin Regulated ALDH1A1 is a Target in Ovarian Cancer Spheroids. Oncogene, 34(18), 2297-2308.

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Culturing Platinum-Resistant Ovarian Cancer Spheres

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The platinum-resistant human ovarian cancer cell line OVCAR-3 was kindly provided by Dr Jiang of West Virginia University. OVCAR-3 cells were cultured in RPMI 1640 medium (Sigma, St Louis, MO, USA) supplemented with 20% US-qualified fetal bovine serum (Invitrogen, Grand Island, NY, USA) at 37 °C with 5% CO₂. To culture spheres of cells, OVCAR-3 cells were harvested and seeded at a density of 2 × 10⁴ cells per well in 6-well ultra-low attachment plates (Corning, NY, USA) in Mammocult complete medium (STEMCELL Technologies, Vancouver, BC, Canada) for 7 days. Spheres were transferred to the next generation by dissociation into single cells with Accutase (STEMCELL Technologies, Vancouver, BC, Canada) and seeded at a density of 2 × 10⁴ cells per well. Third-generation spheroid (SP) cells were used for all the following experiments.

Zhang Y., Chen S., Wei C., Rankin G.O., Ye X, & Chen Y.C. (2018). Dietary compound proanthocyanidins from Chinese bayberry (Myrica rubra Sieb. et Zucc.) leaves attenuate chemotherapy-resistant ovarian cancer stem cell traits via targeting the Wnt/β-catenin signaling pathway and inducing G1 cell cycle arrest. Food & function, 9(1), 525-533.

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