Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA174349

Local Sample ID:	20190525_Indranil_pos_spme_meta5_1
Subject ID:	SU001938
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Female

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Treatment:

Treatment ID:	TR001950
Treatment Summary:	Human breast epithelial MCF10A cells were kindly provided by Prof. Senthil Muthuswamy (Beth Israel Deaconess Medical Center, Harvard Medical School). Cells were cultured in DMEM/F-12 supplemented with 5% Horse serum, EGF 20 ng/mL (Sigma), Insulin 10 μg/mL (Sigma), Hydrocortisone 0.5 mg/mL (Sigma), Cholera toxin 100 ng/mL (Sigma), 100 units/mL Penicillin and 100 μg/mL Streptomycin (HyClone) and grown at 37°C in a humidified incubator with 5% CO2. To induce EMT, cells were stimulated with 10 ng/mL TGF-β1 (Invivogen) and treatments were staggered such that all cells (plates) were harvested at the same time. To minimize cross-contamination (EV & Sec) and promiscuous background signaling (particularly for Phos), cells were cultured in serum-free conditions for 16 hours prior to harvesting. At the time of harvest, conditioned media were first transferred to fresh 50 mL tubes and kept on ice. Cells were washed once with ice-cold PBS and scraped off the plates in ice-cold PBS. Each sample was then distributed into multiple aliquots for multi-omics extractions, centrifuged at 800×g for 5 minutes at 4°C and stored as dry pellets at –80°C. Live cells were imaged in their culture vessels before harvesting using ZOE fluorescent cell imager (Bio-Rad).

Treatment ID:

TR001950

Treatment Summary:

Human breast epithelial MCF10A cells were kindly provided by Prof. Senthil Muthuswamy (Beth Israel Deaconess Medical Center, Harvard Medical School). Cells were cultured in DMEM/F-12 supplemented with 5% Horse serum, EGF 20 ng/mL (Sigma), Insulin 10 μg/mL (Sigma), Hydrocortisone 0.5 mg/mL (Sigma), Cholera toxin 100 ng/mL (Sigma), 100 units/mL Penicillin and 100 μg/mL Streptomycin (HyClone) and grown at 37°C in a humidified incubator with 5% CO2. To induce EMT, cells were stimulated with 10 ng/mL TGF-β1 (Invivogen) and treatments were staggered such that all cells (plates) were harvested at the same time. To minimize cross-contamination (EV & Sec) and promiscuous background signaling (particularly for Phos), cells were cultured in serum-free conditions for 16 hours prior to harvesting. At the time of harvest, conditioned media were first transferred to fresh 50 mL tubes and kept on ice. Cells were washed once with ice-cold PBS and scraped off the plates in ice-cold PBS. Each sample was then distributed into multiple aliquots for multi-omics extractions, centrifuged at 800×g for 5 minutes at 4°C and stored as dry pellets at –80°C. Live cells were imaged in their culture vessels before harvesting using ZOE fluorescent cell imager (Bio-Rad).