Gibco DMEM/F-12, HEPES

3D Cell Culture Media Mouse primary mammary ephitelial cells- organoids

Experiment
3D Cell Culture Media Mouse primary mammary ephitelial cells- organoids
Product
Gibco DMEM/F-12, HEPES from Thermo Fisher Scientific
Manufacturer
Thermo Fisher Scientific

Protocol tips

Upstream tips
Defrost the Matrigel in advance on ice or in fridge
Protocol tips
Use of an eight-well LabTek chamber
Coat the wells with 60μL of a 60% Matrigel (BD Biosciences) solution in BBM

Publication protocol

Mouse organoids were prepared from 13- to 16-wk-old virgin C3H mice following the protocol described by Hirai et al. (1998) with modification. The second through fifth pairs of mammary glands were removed, minced, and incubated for 1 h at 37°C with agitation (500 rpm) with 12.5 mL of Collagenase A medium with Collagenase A at a concentration of 2 mg/mL. The cell suspension was treated as described above except that DNase I was added at 2 U/mL for 1 min. For breastoid and mouse organoid plating, a volume of 60 μL of a 60% Matrigel (BD Biosciences) solution in BBM was added into each well of an eight-well LabTek chamber (Thermo Scientific) and allowed to solidify for 15 min in a 37°C 5% CO2incubator. In a separate tube, 30–40 medium-sized breastoids or mouse organoids per well were added and the volume was brought up to 40 μL using a 50% Matrigel solution in BBM. This mixture was added to the solidified Matigel and allowed to solidify for 15 min in a 37°C 5% CO2 incubator. BBM (350 μL) supplemented with growth factors was then added. EGF (Calbiochem), FGF7 (R&D Systems), AREG (R&D Systems), NRG1-β1 (R&D Systems), and TGFα (Sigma) were used at 5 nM, and FGF2 (R&D Systems) was used at 1 nM unless stated otherwise. The medium was replaced every 2–3 d. U0126 (20 μM) (Sigma) and SL0101 (100 μM) were added with fresh medium every 48 h. Breastoid base medium (BBM) (DMEM/F12 with 15 mM HEPES at pH 7.4 [Invitrogen] [DMEM/F12], 100 μM ethanolamine [Sigma], 1 μg/mL hydrocortisone [Sigma], 10 μg/mL insulin–5.5 μg/mL transferrin–6.7 ng/mL selenium [Invitrogen], 100 U/mL penicillin–100 μg/mL streptomycin [Invitrogen], 2.5 μg/mL Amphotericin B [Sigma], 50 μg/mL gentamicin [Invitrogen]), 5% fetal bovine serum (FBS) (Atlanta Biologicals), and 5% dimethyl sulfoxide.

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Manufacturer protocol

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