EpiCult™-B Mouse Medium Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Use freshly sorted cells	Seed 4,000 cells per well at a density of 8,000 viable cells/mL Use low attachment plates

Upstream tips
Use freshly sorted cells
Protocol tips
Seed 4,000 cells per well at a density of 8,000 viable cells/mL Use low attachment plates

Publication protocol

Freshly sorted basal MMECs from CO- and 4OHT-treated FVB/Tfap2cfl/fl/Krt5-CreERT2 littermate-controlled mice were cultured at 4,000 cells per well at a density of 8,000 viable cells/mL on low attachment plates in EpiCult-B basal medium supplemented with EpiCult-B proliferation supplement, 10 ng/mL epidermal growth factor, 10 ng/mL fibroblast growth factor β, 4 μg/mL, 10% FBS, and 50 μg/mL gentamicin in a low-oxygen incubator at 37°C for 14 days. At 21 days, secondary mammospheres were generated by introduction of trypsin and mechanical disruption of primary mammospheres via gentle aspiration through a 20G syringed needles. Secondary mammospheres were incubated in a low-oxygen incubator at 37°C for 14 days. Total mammosphere count was evaluated by ImageJ software using 40 μm minimum diameter and evidence of 3D structure to differentiate from singular BCs, cellular aggregates, and other debris. Mammosphere formation efficiency was determined by percentage of mammospheres formed per plated basal epithelial cell.

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Manufacturer protocol

Download the product protocol from STEMCELL technologies for EpiCult™-B Mouse Medium Kit below.

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