Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Growth Medium: DMEM/F12 (Merck Millipore, Germany) supplemented with 20% KnockOut™ Serum Replacement (KnockOut™ SR, Thermo Fisher Scientific Inc., MA, USA), 1% penicillin-streptomycin (Sigma-Aldrich; Merck KGaA), 1 mM non-essential amino acid (NEAA; Sigma-Aldrich; Merck KGaA), 0.2 mM 2-mercaptoethanol (Sigma-Aldrich; Merck KGaA), and 10 ng/ml basic fibroblast growth factor (bFGF) (Sigma-Aldrich; Merck KGaA)	Growth medium with the addition of 10 µM ROCK-inhibitor Y-27632 (Sigma-Aldrich; Merck KGaA). Next, cells were left for spontaneous aggregation for 15 days. The medium (hESC medium without bFGF) was changed every second day.

Upstream tips
Growth Medium: DMEM/F12 (Merck Millipore, Germany) supplemented with 20% KnockOut™ Serum Replacement (KnockOut™ SR, Thermo Fisher Scientific Inc., MA, USA), 1% penicillin-streptomycin (Sigma-Aldrich; Merck KGaA), 1 mM non-essential amino acid (NEAA; Sigma-Aldrich; Merck KGaA), 0.2 mM 2-mercaptoethanol (Sigma-Aldrich; Merck KGaA), and 10 ng/ml basic fibroblast growth factor (bFGF) (Sigma-Aldrich; Merck KGaA)
Protocol tips
Growth medium with the addition of 10 µM ROCK-inhibitor Y-27632 (Sigma-Aldrich; Merck KGaA). Next, cells were left for spontaneous aggregation for 15 days. The medium (hESC medium without bFGF) was changed every second day.

Publication protocol

Growth medium with the addition of 10 µM ROCK-inhibitor Y-27632 (Sigma-Aldrich; Merck KGaA). Next, cells were left for spontaneous aggregation for 15 days. The medium (hESC medium without bFGF) was changed every second day.

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Manufacturer protocol

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