pMMECs were derived by harvesting the 4th and 5th mammary glands from 6–12-week-old female transgenic mice with the desired genotype. Glands were incubated in Liberase digestion medium (EpiCult-B Mouse Medium Kit (Stem Cell Technologies, 285 Units CollagenaseType 3 (Worthington), 20mM HEPES (GIBCO), 20 ug/mL Liberase Blendzyme 2 (Roche) and shaken (vertically) at 37°C overnight. The resulting digestion was spun down and resuspended in 3 mls trypsin with EDTA and 1000U DNase and incubated at 37°C for 5 min. LA-7 medium (DMEM-F12 media, 10% FBS, 20 mM HEPES, 10 μg/mL Insulin, 1XL-glutamine, 1X Penicillin-Streptomycin) was added to neutralize the trypsin. Cells were spun down and resuspended in 10U Dispase (Stem Cell Technologies) and 1000U DNase I (Worthington Biochemical) and incubated at 37°C for 5 min. Cells were washed twice with LA-7 medium and the resulting cells were resuspended in EpiCult-B Mouse Medium Kit (Stem Cell Technologies) and seeded onto Cultrex3D-Culture Matrix (Trevigen) coated 6 well plates. For longer term cell growth experiments, pMMECs were seeded on lethally irradiated LA-7 cells and cultured in LA-7 medium. All cells were cultured to 80% confluence then passaged by trypsinization. Cells were tested monthly for mycoplasma using PlasmoTest Kit.
Epicult b mouse medium kit
Manufactured by STEMCELL
Sourced in United States
The EpiCult-B Mouse Medium Kit is a specialized culture medium designed for the expansion and maintenance of mouse mammary epithelial cells. The kit provides the necessary components to support the growth and proliferation of these cell types in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Hepes, by Thermo Fisher Scientific (2 mentions)
Dispase, by STEMCELL (2 mentions)
Collagenase type 3, by Worthington (1 mentions)
Liberase blendzyme 2, by Roche (1 mentions)
Dnase 1, by Worthington (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Countess automated cell counter, by Thermo Fisher Scientific (1 mentions)
Collagenase hyaluronidase, by STEMCELL (1 mentions)
Miltenyi tumor dissociation kit, by Miltenyi Biotec (1 mentions)
Bt474, by American Type Culture Collection (1 mentions)
Mcf 7, by American Type Culture Collection (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by STEMCELL (1 mentions)
Dnase, by Worthington (1 mentions)
4 protocols using epicult b mouse medium kit
1
Isolation and Culture of pMMECs
2
Single-cell Isolation from Mammary Tissue
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The mammary glands for prepuberty and DCIS were placed in 10 ml of a digestion medium containing EpiCult™-B Mouse Medium Kit (#05610, StemCell Technologies), Collagenase/Hyaluronidase (#07912, StemCell Technologies), and 1% penicillin-streptomycin (Gibco). The mammary gland was digested overnight in a thermocycler maintained at 37 °C with continuous rotation. The C3(1)-Tag tumors were digested with the Miltenyi tumor dissociation kit (#130-096-730, Miltenyi Biotech) under a gentle agitation setting. The cell pellets retrieved from these suspensions were treated with a 1:4 solution of hanks balanced salt solution (HBSS) and ammonium chloride to remove the RBCs. After RBC removal, the cell suspensions were trypsinized with 0.05% Trypsin and a mix of Dispase and DNAse. A portion of this cell suspension was stained with trypan blue and counted using the Countess Automated Cell Counter (Invitrogen). Based on the counting, the cells were diluted to the appropriate cell stock concentration for running on the 10× chromium machine. Based on the 10× genomics pre-defined cell stock concentrations, each experiment was run to retrieve ~5000 cells after the single cell experiment.
3
Breast Cancer Cell Line Culture
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Breast cancer cell lines MCF-7, BT474 and T47D were purchased from ATCC and SUM149 were obtained from Asterand. All cell lines were cultured according to the recommends from ATCC or Asterand. Primary cells used in the study were derived from digested tumor tissues and cultured with EpiCult™-B Mouse Medium Kit (#05610, STEMCELL, USA) under directions.
4
Isolation of Primary Mouse Mammary Epithelial Cells
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pMMECs were derived by collecting the fourth and fifth mammary glands from 8–12-week-old virgin female transgenic mice with the desired genotype. pMMECs were isolated without performing randomization. Glands were incubated in Liberase digestion medium ((EpiCult-B Mouse Medium kit, StemCell Technologies, 05610), 150 U ml–1 collagenase type 3 (Worthington, LS004182), 20 mM HEPES (Thermo Fisher Scientific, 15630106) and 20 µg ml–1 Liberase Blendzyme 2 (Roche, 11988425001)) and shaken vertically at 37 °C overnight. The resulting digestion was spun down and resuspended in trypsin (Gibco, 25200056) with DNase I (Worthington, LS002060) and incubated at 37 °C for 5 min. DMEM with 5% FBS was added to neutralize the trypsin. Cells were spun down and resuspended in dispase (Stem Cell Technologies, 07913) and DNase I and incubated at 37 °C for 5 min. Cells were washed twice with DMEM, and the resulting cells were resuspended in Epi-Cult medium and seeded onto plates coated with Cultrex 3D culture matrix rat collagen I (Fisher Scientific, 3447-020-01).
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