Corning® 500 mL Iscove’s Modification of DMEM

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Defrost the Matrigel in advance on ice or in fridge	lnduce branching of lung bud organoids using Matrigel To prepare the base of Serum-free differentiation (SFD) media, you need to use IMDM/Ham’s F12 (3:1) with supplements

Upstream tips
Defrost the Matrigel in advance on ice or in fridge
Protocol tips
lnduce branching of lung bud organoids using Matrigel To prepare the base of Serum-free differentiation (SFD) media, you need to use IMDM/Ham’s F12 (3:1) with supplements

Publication protocol

hPSC maintenance media consisted of DMEM/F12 (1:1) supplemented with 20% knockout serum replacement, 0.1 mM β-mercaptoethanol, Primocin, and 20 ng/ml FGF-2. Serum-free differentiation (SFD) media consisted of IMDM/Ham’s F12 (3:1) supplemented with N2, B27, 0.05% bovine serum albumin, 1% penicillin-streptomycin, 50 µg/ml ascorbic acid, 2mM Glutamax, 0.4 µM monothioglycerol and different growth factor cocktails as indicated in Supplementary Table 2. Formation of lung bud organoids: At the end of anterior foregut endoderm induction, cells were treated with Ventralization media (Branching media) for 48 h and three-dimensional clump formation was observed. The clumps were then suspended by gently pipetting around the wells. The suspended clumps are called lung bud organoids (LBOs) hereafter. LBOs were maintained in non-tissue culture treated multiple-well plates submerged in Branching media and were fed every other day until d20–d25. Branching morphogenesis in Matrigel: The d20-d25 LBOs were embedded in 100% Matrigel in 24-well transwell inserts and incubated in incubator until the Matrigel solidified. Branching media were added to the well, after which the transwell was inserted, branching media added into the transwell insert as well. Media were changed every other day. A step-by-step protocol describing the generation of LBOs and LBO-derived branching colonies in Matrigel can be found at Nature Protocol Exchange41.

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Manufacturer protocol

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