Gibco™KnockOut™ DMEM

Protocol tips

Upstream tips	Protocol tips	Downstream tips
growth media (80% KO-DMEM, 20% Knock-out Serum Replacer (KSR), 1% non-essential amino acids, 2 mM Glutamax, 0.1 mM ß-Mercaptoethanol, and 1% penicillin and streptomycin all Life Technologies), supplemented with either 10 ng/ml (Shef hES and NAS2 hiPS) or 20 ng/ml (MSU0001) FGF2 (Peprotech).	Colonies were chopped into uniform 150 μm pieces using a McIlwain tissue chopper (Mickle Engineering, Gomshall, U.K.), and resuspended in growth media (described above), without FGF2 but supplemented with 10 μM of the ROCK inhibitor Y27632 (Tocris)	Colony pieces formed spheres and differentiated into EBs over 4 subsequent days, after which they were transferred into modified chemically defined media (CDM) (Joannides et al., 2007), (BSA was substituted with 400 μg/ml Albumax-II, Life Technologies). CDM was supplemented with 10 μM Y27632 (Tocris) and 20 μM of the Nodal/TGF-β signaling inhibitor SB431542 (Tocris)

Upstream tips
growth media (80% KO-DMEM, 20% Knock-out Serum Replacer (KSR), 1% non-essential amino acids, 2 mM Glutamax, 0.1 mM ß-Mercaptoethanol, and 1% penicillin and streptomycin all Life Technologies), supplemented with either 10 ng/ml (Shef hES and NAS2 hiPS) or 20 ng/ml (MSU0001) FGF2 (Peprotech).
Protocol tips
Colonies were chopped into uniform 150 μm pieces using a McIlwain tissue chopper (Mickle Engineering, Gomshall, U.K.), and resuspended in growth media (described above), without FGF2 but supplemented with 10 μM of the ROCK inhibitor Y27632 (Tocris)
Downstream tips
Colony pieces formed spheres and differentiated into EBs over 4 subsequent days, after which they were transferred into modified chemically defined media (CDM) (Joannides et al., 2007), (BSA was substituted with 400 μg/ml Albumax-II, Life Technologies). CDM was supplemented with 10 μM Y27632 (Tocris) and 20 μM of the Nodal/TGF-β signaling inhibitor SB431542 (Tocris)

Publication protocol

Colonies were chopped into uniform 150 μm pieces using a McIlwain tissue chopper (Mickle Engineering, Gomshall, U.K.), and resuspended in growth media (described above), without FGF2 but supplemented with 10 μM of the ROCK inhibitor Y27632 (Tocris)

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for Gibco™KnockOut™ DMEM below.

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