This is a general outline of the procedure we've used:
1. Suspended 600,000 cells in 6 mL of alpha-MEM supplemented with ~1% DMSO, 10%FBS, and 1% Pen/Strp (antiobiotic).
2. Cultured them for 4 days in bacterial petri dishes. (On the third day of suspension culture, the media was "changed" by doubling the appropriate media volume)
3. After 4 days the suspended aggregates were re-plated onto 12 well plates and cultured without DMSO for either 2, 4, or 6 more days, with the media being changed every two days.
We didn't see beating at each time point and couldn't make out any changes in cell morphology. Does anyone have any suggestions considering changes that should be made to the protocol? Do you have a protocol that's been successful, that you think I should look into? Anything would help, really. Thanks a lot!
Did those aggregates stick down into wells? I think they should be big enough and must keep them aggregated for proper diff. We use the very same protocol to get neuronal cultures with RA treatment. So pipette those aggregates very carefully.
To me, the critical factors are the cells themselves and the serum used.
If you cannot get your cells to beat, change to another lot, or get it from another lab. By the way are you using P19CL6? It is a better clone for cardiac differentiation.
Try another lot of serum as well. You may also want to try the hanging drop method. Search for my "Sox17 is essential for cardiac mesoderm..." paper. I used ESCs but the handing drop method is the same.
From http://www.ijdb.ehu.es/web/paper.php?doi=15005574:
Hanging drops containing 800 cells in a volume of 20 µL (DMEM/Hams F12 (or just DMEM) + GlutaMAX or L-Glut, supplemented with 7.5% FCS, non-essential aa (optional), and +/- 1% DMSO) were plated onto the lid of bacterial grade culture dishes and incubated at 37°C for 4 days (Mummery et al., 1991; Slager et al., 1993). The base of the each culture dish was filled with PBS to prevent the drops from evaporating. On
the 5th day, the aggregates were collected from the hanging drops and
plated in α-MEM growth media with 10% FCS (no DMSO) on glass cover slips or in
TC treated plastics. The media was then refreshed every 48 hours and the
aggregates were examined every day for the presence of beating muscle.
The alpha-MEM works better for monolayer than hanging drop I think. And you only need 5% serum or less for P19Cl6 cells since they already proliferate like crazy.
And yes, serum batches tend to differ a lot so test a couple from different suppliers and buy in bulk when you've found the right one.
- Badges
- Science method