Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Process tumor specimens 1h of surgery	Use ultralow attachment plates Change media every third day

Upstream tips
Process tumor specimens 1h of surgery
Protocol tips
Use ultralow attachment plates Change media every third day

Publication protocol

Tumor specimens were processed within 1 h of surgery. Each sample was minced and digested at 370C, for 2 to 4 h in a solution of type IV collagenase, 1 mg mL-1 (Sigma), containing 40 mg mL-1 of bovine serum albumin, 2 mg mL-1 of glucose, 100 units mL-1 of penicillin and 100 mg mL-1 of streptomycin (P+S; Sigma), 50 mg L-1 of gentamicin (Sigma), and 1.25 mg/L of Fungizone (Life Technologies). After incubation, the samples were extensively rinsed with PBS and suspended in culture media. Cells were subsequently subjected to centrifugation at 40X g for generation of epithelial breast – enriched fraction. Single cells were plated in ultralow attachment plates (Corning) in low densities. Cells were grown in a serum-free DMEM/F12 (Sigma) supplemented with 2% B27 (Invitrogen), 20 ng/ml EGF (Peprotech) and 1% P+S. Media was changed every third day.


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Manufacturer protocol

Download the product protocol from Sigma-Aldrich for Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham below.

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