Tina:Cardiomyocyte differentiation protocols: Difference between revisions
>TinaLo (Created page with "==Monolayer-directed cardiomyocyte differentiation protocol== ===Christian Metallo Lab Protocol=== 1. Prepare cells on Matrigel-coated plates. Wait until cells reach 100% conf...") |
>TinaLo |
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Line 5: | Line 5: | ||
2. [Day 0] | 2. [Day 0] | ||
Aspirate culture medium. Add 2 ml RPMI/ | 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. | 3. [Day 1] Each day is an exact 24 hrs period. | ||
Aspirate medium. Add 2 ml RPMI/ | Aspirate medium. Add 2 ml RPMI/B-27-A medium per well of a 12-well plate. No CHIR99021 required. | ||
4. [Day 2] | 4. [Day 2] | ||
Add DMK stock into culture medium (final conc. 0.5 mM DMK). Do not change the | 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. |
Latest revision as of 00:53, 3 April 2013
Monolayer-directed cardiomyocyte differentiation protocol[edit]
Christian Metallo Lab Protocol[edit]
1. Prepare cells on Matrigel-coated plates. Wait until cells reach 100% confluency.
2. [Day 0]
Prepare RPMI/B-27-A medium.
Component | Volume |
RPMI 1640 medium, without Gln | 49 ml |
B-27 supplement minus insulin | 1 ml |
Total | 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.
Component | 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 |
Total | 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).
Component | Volume |
RPMI 1640 medium, without Gln | 49 ml |
B-27 serum free supplement | 1 ml |
Total | 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.