MammoCult™ Human Medium Kit

Protocol tips

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	Incubation of mammospheres under hypoxia Passages of mammospheres every 4 days Count of mammospheres at least 50 μm diameter

Protocol tips
Incubation of mammospheres under hypoxia Passages of mammospheres every 4 days Count of mammospheres at least 50 μm diameter

Publication protocol

For the mammosphere culture of breast tumor cells from clinical patients, the tumor tissues were minced and digested with 1 mg/mL of type 1 collagenase (Sigma) at 37 °C for 30 min to prepare single-cell suspensions. Mammosphere cells were grown in Complete Mammm Cult Medium (Stem Cell Technologies) or a serum-free Dulbecco's Modified Eagle's medium (DMEM)/F12 medium supplemented with B27 (Invitrogen), 20 ng/mL EGF, 20 ng/mL bFGF, 0.4% BSA, 2 μg/mL heparin, and 5 μg/mL insulin. The incubation of mammospheres was carried out under hypoxia (1% O2). Mammospheres were passaged every 4 days. The numbers of secondary-generation spheres (at least 50 μm diameter) were counted using an OLYMPUS IX70 microscope (Tokyo, Japan) fitted with graticule at ×100 magnification. Calculate the percentage of mammosphere forming efficiency (MFE). The average size of the randomly selected mammospheres (N = 30) was calculated. The percentage of MFE (%) from primary to tertiary generation indicated the self-renewal capacity of each kind of mammospheres. Mammosphere cell growth was determined by cell numbers contained within each generation of mammospheres. All experiments were performed at least three times.

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Manufacturer protocol

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

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