Tina:Cardiomyocyte differentiation protocols: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>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
 
Line 5: Line 5:
2. [Day 0]
2. [Day 0]


Aspirate culture medium. Add 2 ml RPMI/B27-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/B27-A medium (final conc. 12 uM CHIR99021).
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/B27-A medium per well of a 12-well plate. No CHIR99021 required.
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 used RPMI/B27-A medium.
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.