Mammary tumor epithelial cells (MECs) were isolated from MMTV-Wnt1 or MMTV-Wnt1/dnIGF-1R mice similarly to our prior study [1 (link)]. Whole tumors were excised and dissociated with the gentleMACs tissue dissociator (130–093-235, protocol m_TDK2) and mouse specific tumor dissociation kit (Miltenyi, 130–096-730). Organoids that retain basement membrane attachments were trypsinized (0.05% Trypsin-EDTA, Gibco) and filtered with a 40-μm cell strainer (BD Biosciences) to isolate a single cell suspension of dissociated tumor MECs. Isolated tumor MECs were counted using a hemocytometer prior to flow cytometry or sorting.
Mammary tumor immune cells were isolated from tumors as described previously [23 ]. Whole tumors were excised, minced, and digested with Collagenase-I (10 U/ml), Collagenase-IV (400 U/ml; Worthington), and DNase-1 (30 mg/ml; Sigma Aldrich) for 25 min at 37 °C. Cells from digested tumors were filtered with a 70-μm cell strainer (BD Biosciences) and pelleted. Red blood cells were lysed with an erythrocyte lysis buffer (150 mM Ammonium chloride, 1 mM Potassium bicarbonate, 130 μM EDTA, pH 7.2) for 2 min, filtered with a 70-μm cell strainer (BD Biosciences) and pelleted. Isolated immune cells were counted using a hemocytometer prior to flow cytometry or magnetic bead sorting.
Mammary tumor immune cells were isolated from tumors as described previously [23 ]. Whole tumors were excised, minced, and digested with Collagenase-I (10 U/ml), Collagenase-IV (400 U/ml; Worthington), and DNase-1 (30 mg/ml; Sigma Aldrich) for 25 min at 37 °C. Cells from digested tumors were filtered with a 70-μm cell strainer (BD Biosciences) and pelleted. Red blood cells were lysed with an erythrocyte lysis buffer (150 mM Ammonium chloride, 1 mM Potassium bicarbonate, 130 μM EDTA, pH 7.2) for 2 min, filtered with a 70-μm cell strainer (BD Biosciences) and pelleted. Isolated immune cells were counted using a hemocytometer prior to flow cytometry or magnetic bead sorting.
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