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Tina:Cardiomyocyte differentiation in H9 ESCs (CDH9)
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==Experiment #1 (CDH9_E1_T1)== *Study Purpose: Try 2 different induction medium and 2 different cell density during differentiation and see how it works on H9 ESCs directed cardiomyocyte differentiation. *Date: 4/10/2013 ~ 5/15/2013 *Cells: H9 ESC line, Brainbow Clone #3 (H9Bb3). Cells seeded on 4/10. (1:6 dilution from the second passage of H9 ESCs under a feeder free culture system) OP9 cells were used as a negative control. *Protocol used: Christian Metallo Lab protocol. *Two induction medium used: Gln+ induction medium,DMK induction medium *Two cell density used: 100%, 80% confluency. '''12-well plate #1''' {| {{table}} border=1 | align="center" style="background:#f0f0f0;"|100% confluency | align="center" style="background:#f0f0f0;"|'''1''' | align="center" style="background:#f0f0f0;"|'''2''' | align="center" style="background:#f0f0f0;"|'''3''' | align="center" style="background:#f0f0f0;"|'''4''' |- | align="center" style="background:#f0f0f0;"|A||Gln, repeat 1||Gln, repeat 2||DMK, repeat 1||DMK, repeat 2 |- | align="center" style="background:#f0f0f0;"|B||Gln, repeat 3||Gln, repeat 4||DMK, repeat 3||DMK, repeat 4 |- | align="center" style="background:#f0f0f0;"|C||Negative ctrl, repeat 1||Negative ctrl, repeat 2||Negative ctrl, repeat 3||Negative ctrl, repeat 4 |- |} '''12-well plate #2''' {| {{table}} border=1 | align="center" style="background:#f0f0f0;"| 80% confluency | align="center" style="background:#f0f0f0;"|'''1''' | align="center" style="background:#f0f0f0;"|'''2''' | align="center" style="background:#f0f0f0;"|'''3''' | align="center" style="background:#f0f0f0;"|'''4''' |- | align="center" style="background:#f0f0f0;"|A||Gln, repeat 1||Gln, repeat 2||DMK, repeat 1||DMK, repeat 2 |- |} *Induction: 4/16 (Day 0). Cells reached 100% and 80% confluency, respectively. Aspirate mTeSR1 medium, wash once with PBS, add Gln+ and DMK induction medium accordingly. Pre-mix CHIR99021 (and DMK, for DMK group only) with culture media, then added to each well. *4/17 (Day 1). Change medium (pre-mix DMK). Observed lots of cell death. Might due to the extremely high cell density and the sudden withdrawal of serum in differentiation medium. *4/18 (Day 2). Still, lots of dead cells. Add 5 ul DMK to DMK group. *4/19 (Day 3). Shake wells before change media to remove dead cells. Most of them has been removed after that. Change to combined medium. *4/20~4/22. Change medium accordingly. Cells were still dying. But many survived also. *4/23 (Day 7). Change to Insulin+ medium. Start to change medium every 3 days from now on. *4/26 (Day 10). Observed one beating colony in 80% confluency, Gln+ group (Plate#2, A1). No signs of any other beating cells in other wells yet. *Pictures:
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