Gibco DMEM/F-12, HEPES

Protocol tips

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

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

Breastoid purification: Human breast tissue from reduction mammoplasty or from the normal margin that was removed during breast cancer surgery was collected as waste tissue with institutional review board approval. A list of the age and race for each of the patient samples used in this study is provided (Supplemental Table 1). The tissue was weighed and minced with scissors into 1-mm3 portions. A portion was taken for fixing in 10% formalin (Protocol) for 2 d and then placed in 70% ethanol. The fixed samples were paraffin-embedded and 5-μm sections were prepared by the University of Virginia Research Histology Core. Tissue was sometimes cryopreserved by adding an equivalent weight of 90% 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. The tissue was thawed at 37°C and placed in prewarmed DMEM/F12 before proceeding. Approximately 15 g of fresh or thawed tissue was digested for 18–21 h at 37°C in a 5% CO2 incubator with sterile 25 mL of Collagenase A medium (DMEM/F12, 1 mg/mL Collagenase A [Roche], 1 μg/mL insulin [Sigma], 600 U/μL Nystatin (Sigma), 100 U/mL penicillin–100 μg/mL streptomycin [Invitrogen]). If the sample was very fibrous, additional Collagenase A medium was added at 16–17 h. The digested material was centrifuged at 180g for 5 min and the solubilized material was decanted from the pellet containing the breastoids. The pellet was washed in 5 mL of prewarmed DMEM/F12 medium. The pellet was resuspended in 5 mL of prewarmed DMEM/F12 with DNase I (1000 U/mL) (Sigma) for 3–5 min at 37°C in a 5% CO2 incubator. FBS (0.5 mL) was added and the suspension was centrifuged at 180g for 10 min. The pellet was resuspended in 9 mL of phosphate-buffered saline (PBS) with 5% FBS and centrifuged at 350g for 15 sec. This wash step was repeated six times. Any fibrous tissue was removed manually. The pellet was resuspended in 9 mL of DMEM/F12 and centrifuged at 350g for 15 sec. The pellet was resuspended in 1 mL of BBM. An aliquot was removed and the number of breastoids was counted.
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.


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