DMEM/F-12

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Defrost the Matrigel in advance on ice or in fridge	Harvest organoids after 4–7 days of growth

Upstream tips
Defrost the Matrigel in advance on ice or in fridge
Protocol tips
Harvest organoids after 4–7 days of growth

Publication protocol

Organoids Derived From Gastric Cancer Tissue: Tumor tissue was obtained from patients undergoing surgical resection for gastric cancer (IRB protocol number: 2015-5537, University of Cincinnati and H-35094, Baylor College of Medicine). Tumor organoids were generated as previously described.30 Briefly, tumor tissue was washed well in DPBS without Ca2+ and Mg2+ supplemented with antibiotics and minced into small pieces. Tumor fragments were placed in pre-warmed stripping buffer: Hank’s balanced salt solution, ethylenediamine tetraacetic acid (5 mmol/L), HEPES (25 mmol/L), 10% fetal calf serum. Fragments were incubated for 10 minutes in a shaking incubator at 37°C. The fragments were supplied fresh stripping buffer and incubated for an additional 5 minutes in a shaking incubator at 37°C. The tissue fragments were washed with Hank’s balanced salt solution twice, and pre-warmed incubation buffer (RPMI supplemented with collagenase [1.5 mg/mL] and hyaluronidase [20 μg/mL]) was added to the fragments and incubated at 37°C for 30 minutes in a shaking incubator. The digest was diluted with 20 mL DPBS without Ca2+ and Mg2+ supplemented with antibiotics and filtered through a 70-μm filter. The cells were centrifuged at 1200 rpm for 5 minutes and embedded into Matrigel supplemented with gastric growth medium as described above.

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for DMEM/F-12 below.

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