After lactate selection and two days of recovery, hiPSC-CMs were dissociated with 10 μm/mL collagenase A and B (Roche) for 30 min, followed by Accutase for 10 min, spun down at 1000 RPM for 5 min. 5 × 106 cells were then mixed with 100 μL of RPMI 1640/B27 (+) complete, 5 μM Y-27632 (ROCK Inhibitor; STEMCELL Technologies) and 70 μL of GFR-Matrigel on ice. hiPSCs were dissociated with dispase for 8 min and spun down at 200 RPM for 3 min. Then, 5 × 106 cells were mixed with 100 μL of media (hESC), 5 μM Y-27632 and 70 μL of GFR-Matrigel on ice, and then subcutaneously injected into 8- to 12-week-old nonobese diabetic SCID-γ mice (Taconic). Tumors were collected between 6 and 8 weeks after the cell injection.
Collagenase a and b
Manufactured by Roche
Collagenase A and B are enzymes used for the digestion and dissociation of collagen, a major structural component of the extracellular matrix. They are used in various cell and tissue culture applications, such as the isolation and purification of cells from tissues.
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Lab products found in correlation
2 protocols using collagenase a and b
1
Subcutaneous Transplantation of hiPSC-CMs in Mice
2
Cardiac Differentiation of hPSCs Using Small Molecules
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Cardiac differentiation of hPSCs was induced using small molecules as previously described (Atmanli et al., 2014 (link)). Briefly, when hPSCs achieved confluency, cells were treated with CHIR99021 (Stemgent) in RPMI (Thermo Fisher) supplemented with Gem21 NeuroPlex without insulin (Gemini Bio Products) for 24 hr (from day 0 to day 1). The medium was replaced with RPMI/G21-insulin at day 1. The cells were then treated with IWP4 (Stemgent) in RPMI/G21-insulin at day 3 and the medium was refreshed on day 5 with RPMI/G21-insulin. Cells were maintained in RPMI supplemented with Gem21 NeuroPlex (Gemini Bio Products) starting from day 7, with the medium changed every 3 days. Ascorbic acid at 50 μg/ml was added to media until onset of beating. Beating clusters were seen starting day 6 of differentiation. Metabolic selection of cardiac myocytes was performed for 3 days by incubating cells in media without glucose but supplemented with 5 mM sodium DL-lactate.
Cardiac myocytes were harvested by treating the cells with Collagenase A and B (Roche) for 5 min first and then TrypLe Express (Thermo Fisher) for another 5 min. Cells were plated onto Matrigel-coated 96-well plates with a No. 1.5 glass bottom or polydimethylsiloxane-coated glass dishes.
Cardiac myocytes were harvested by treating the cells with Collagenase A and B (Roche) for 5 min first and then TrypLe Express (Thermo Fisher) for another 5 min. Cells were plated onto Matrigel-coated 96-well plates with a No. 1.5 glass bottom or polydimethylsiloxane-coated glass dishes.
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