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Tina:Cardiomyocyte differentiation protocols
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===Christian Metallo Lab Protocol=== 1. Prepare cells on Matrigel-coated plates. Wait until cells reach 100% confluency. 2. [Day 0] Prepare RPMI/B-27-A medium. {| {{table}} border=1 | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | RPMI 1640 medium, without Gln||49 ml |- | B-27 supplement minus insulin||1 ml |- | style="background:#f0f0f0;"|Total | style="background:#f0f0f0;"|50 mL |} Aspirate culture medium. Add 2 ml RPMI/B-27-A medium per well of a 12-well plate (or 1 ml medium per well of a 24-well plate). Add CHIR99021 stock into RPMI/B-27-A medium (final conc. 12 uM CHIR99021). 3. [Day 1] Each day is an exact 24 hrs period. Aspirate medium. Add 2 ml RPMI/B-27-A medium per well of a 12-well plate. No CHIR99021 required. 4. [Day 2] Add DMK stock into culture medium (final conc. 0.5 mM DMK). Do not change the basal RPMI/B-27-A medium. 5. [Day 3] Prepare Combined Medium. {| {{table}} border=1 | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | Spent medium||1 ml |- | Fresh RPMI/B-27A with 0.5 mM DMK)||1 ml |- | IWP2||2 ul of 5mM stock; final conc. 5uM |- | style="background:#f0f0f0;"|Total | style="background:#f0f0f0;"|2 mL |} Shake the plate back and forth gently, aspirate the remaining medium and cell debris. Add 2ml Combined Medium per well. 6. [Day 4] Add DMK stock into culture medium (final conc. 0.5 mM DMK). Do not change the basal Combined Medium. 7. [Day 5] Aspirate medium. Add 2 ml RPMI/B-27-A medium per well. No other supplements required. 8. [Day 6] Add DMK stock into culture medium (final conc. 0.5 mM DMK). Do not change the basal RPMI/B-27-A medium. 9. [Day 7] Prepare RPMI/B-27-B medium (Insulin Plus formula). {| {{table}} border=1 | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | RPMI 1640 medium, without Gln||49 ml |- | B-27 serum free supplement||1 ml |- | style="background:#f0f0f0;"|Total | style="background:#f0f0f0;"|50 mL |} Aspirate medium. Add 2 ml RPMI/B-27-B medium per well. 10. Change new RPMI/B-27-B medium every 3 days.Robust spontaneous contraction should occur by Day 12-15 (as early as Day 9). Cells can maintain its beating phenotype for months.
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