MammoCult™ Human Medium Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Use of sorted basal and luminal epithelial population	Seed 5,000–20,000 cells per well for basal cells and 2,000–10,000 cells per well for luminal cells Use of ultralow attachment 96-well plates After 6 days, count the spheres with diameter greater than 30 μm

Upstream tips
Use of sorted basal and luminal epithelial population
Protocol tips
Seed 5,000–20,000 cells per well for basal cells and 2,000–10,000 cells per well for luminal cells Use of ultralow attachment 96-well plates After 6 days, count the spheres with diameter greater than 30 μm

Publication protocol

Digest the human breast tissue using collagenase/hyaluronidase (tissue with less than 2g is digested in 3 mL digestion medium). Rapiddly, wash the digested tissue with cold PBS in a 60 mm culture dish placed on ice and mince with a scissor. Remove fat and lyse the red cells. Prepare single cells adding trypsin-EDTA and dispase treatment. This whole procedure usually takes 2hr. Proceed to Flow Cytometry sorting of basal mammary epithelial cells (Lin−/CD49fhigh/EpCAM−), luminal progenitor cells (Lin−/CD49flow/EpCAM+), and mature luminal cells (Lin−/CD49f−/EpCAM+). Resuspend the cell pellet in complete MammoCult™ Human Medium and plate cells on 96 well ultra-low attachment plates at 5,000–20,000 cells per well for basal cells and 2,000–10,000 cells per well for luminal cells respectively. A volume of 100 μL MammoCult™ Human Medium is used per well. Feed the cells every 3 days by adding another 50 μL medium each time. Complete MammoCult™ Human Medium: MammoCult™ Basal Medium (Human, CAT #05621) 9 mL MammoCult™ Proliferation Supplement (Human, CAT #05622) 1 mL Heparin, 0.2 % 20 μL, Hydrocortisone, 2.5 mg/mL 1.9 μL, Penicillin/Streptomycin 100 μL

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Manufacturer protocol

Download the product protocol from STEMCELL technologies for MammoCult™ Human Medium Kit below.

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