STEMdiff™ Trilineage Differentiation Kit

Protocol tips

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	Plate cells onto Matrigel (Corning) and treat with endoderm or mesoderm differentiation media for 5 days or ectoderm differentiation media for 7 days.

Protocol tips
Plate cells onto Matrigel (Corning) and treat with endoderm or mesoderm differentiation media for 5 days or ectoderm differentiation media for 7 days.

Publication protocol

Primed induced pluripotent stem cells (UMN PCBC16iPS) [5] to be used for naive cell derivation were cultured for at least two passages in mTeSR1 medium (STEMCELL Technologies) in either normoxic or hypoxic conditions. Prime induced pluripotent stem cells (iPSCs) were treated with Gentle Cell Dissociation Buffer (STEMCELL Technologies) for 5 min at room temperature and removed from the plate with a 5 mL pipette in 1 mL TeSR1 medium. Aggregates of 250 prime iPSCs were plated in 1 mL mTeSR1 (STEMCELL Technologies) per well of a 12-well tissue culture treated plate (Corning) coated with 5 μg/mL full-length vitronectin (PeproTech) [6]. The next day, cells were transferred to hypoxic conditions and switched to RSeT medium (STEMCELL Technologies) culture conditions. Cells were monitored for compaction and defined colony edges. When naive iPSC colonies reached 250 mm in diameter, the well was passaged. For passaging, cells were washed 1× with phosphate-buffered saline (PBS)–Ca2+/-Mg2+ followed by addition of 250 μL TrypLE Express (Thermo Scientific) for 3 min at 37°C in hypoxic conditions. TrypLE was neutralized with 750 μL RSeT supplemented with 10 μM Rho-associated kinase inhibitor Y-27632 (BD Biosciences). Cells were agitated 3× using a p1000 pipet tip to achieve 10–12 cell clumps. After collection, cells were spun at 300 g for 5 min. Cells were resuspended in 120 μL of RSeT medium supplemented with 10 μM Y-27632 by pipetting 6× with a p200 pipet tip to break cells into clumps of 2–3 cells. Cells were plated in dilutions of 1:3, 1:4, 1:8, and 1:10 to ensure optimal plating density.

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for STEMdiff™ Trilineage Differentiation Kit below.

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