DMEM/F-12, no phenol red

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	Seed 3000 cells/well (MCF7) Use of pHEMA (Sigma) -coated 6-well plates Score of mammospheres > 50μm

Protocol tips
Seed 3000 cells/well (MCF7) Use of pHEMA (Sigma) -coated 6-well plates Score of mammospheres > 50μm

Publication protocol

MCF7 cells were trypsinised and passed through a 25G needle to generate a single cell suspension. Cells were plated onto pHEMA (Sigma) -coated 6-well plates at a density of 3000 cells/well for MCF7. Cells were cultured in DMEM/F12 (Thermo Scientific 21041025) supplemented with B27 (Thermo Scientific 12587010), 20ng/ml EGF (Miltenyi) and 5U/ml penicillin and 1mg/ml streptomycin. Mammospheres > 50μm were scored using a graticle. To assess self- renewal capabilities, mammospheres were harvested and disaggregated into single cell suspension as previously. Cells were cultured in pHEMA-coated 6-well plates for a further five days prior to scoring. For mammosphere assays from xenografts, tumours were dissociated using the Miltenyi Tumour Cell Dissociation kit according to manufacturer’s instructions. Cells were plated at 5000 cells/well and scored for both primary and secondary mammospheres and expressed as mammosphere forming unit (MFU).

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