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Found 2 matching solutions for this experiment
Upstream tips |
Protocol tips |
Downstream tips |
Prior to differentiation, it is essential to ensure 80–90% of cells are undifferentiated and cultures are 70–80% confluent. Prior to dissociation cells are treated with 10 μM ROCK inhibitor (Y-27632) to reduce dissociation-induced apoptosis |
E8 media supplemented with 10 μM Y-27632 |
Cells are then treated with 10 μM CHIR99021 supplemented E6 media—E8 media lacking the pluripotency factors FGF2 and TGF-β1—to simulate Wnt signaling during gastrulation |
Upstream tips |
Prior to differentiation, it is essential to ensure 80–90% of cells are undifferentiated and cultures are 70–80% confluent. Prior to dissociation cells are treated with 10 μM ROCK inhibitor (Y-27632) to reduce dissociation-induced apoptosis |
Protocol tips |
E8 media supplemented with 10 μM Y-27632 |
Downstream tips |
Cells are then treated with 10 μM CHIR99021 supplemented E6 media—E8 media lacking the pluripotency factors FGF2 and TGF-β1—to simulate Wnt signaling during gastrulation |
Upstream tips |
Protocol tips |
Downstream tips |
cells infected with Tet-on lentivirus |
E8 medium supplemented with Y27632 (5 µM, Tocris) at 3.3 × 104 cells/cm2. The following day, E8 media was replaced with E6 media and cells were cultured for 2 days supplemented with CHIR99021 (10 µM, Selleck Chemical), after which CHIR99021 was removed and E6 media supplemented with 1 µg/mL Dox (Sigma) for 18 days until GFP+ induced myogenic progenitor cells (iMPCs) were sorted by FACS as described below. During differentiation of iMPCs, 10 ng/mL bFGF (R&D) was added starting at day 5 to enhance proliferation of GFP+ cells. |
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Upstream tips |
cells infected with Tet-on lentivirus |
Protocol tips |
E8 medium supplemented with Y27632 (5 µM, Tocris) at 3.3 × 104 cells/cm2. The following day, E8 media was replaced with E6 media and cells were cultured for 2 days supplemented with CHIR99021 (10 µM, Selleck Chemical), after which CHIR99021 was removed and E6 media supplemented with 1 µg/mL Dox (Sigma) for 18 days until GFP+ induced myogenic progenitor cells (iMPCs) were sorted by FACS as described below. During differentiation of iMPCs, 10 ng/mL bFGF (R&D) was added starting at day 5 to enhance proliferation of GFP+ cells. |
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