1. Stem Cell culturing

2. PLA Experiment #1 (C2C12 cells)
Per GROUP/TEAM
Aspirate off the media
IMMEDIATELY add 2ml of trypsin to flask, rock and remove IMMEDIATELY
IMMEDIATELY add 1ml of trypsin, allow to sit for 4 min MAX
Add 5ml of 10% media to the flask to stop the reaction
Rinse the scaffold with 1ml of media, remove
Add 1ml of fresh media to 4 wells (including the scaffold well)
Transfer the cells = 1ml to scaffold well, and then 0.5ml, 1ml and 2 ml (see diagram)
Scaffold 1
0.5 2

3. Differentiaton Experiment (C2C12 cells)
Per PERSON (4 plates each)
Aspirate off the media
IMMEDIATELY add 2ml of trypsin to flask, rock and remove IMMEDIATELY
IMMEDIATELY Add 1ml of trypsin, allow to sit for 4 min MAX
Add 7ml of 10% media to the flask to stop the reaction
Add 3ml of fresh media to each plate
Transfer the cells = 3ml. 2ml, 1ml, 1ml (see diagram)
3ml
2ml
1ml

4. Scratch Test(3T3 cells) Per TABLE Aspirate off the media
IMMEDIATELY add 2ml of trypsin to flask, rock and remove IMMEDIATELY
IMMEDIATELY add 1ml of trypsin, allow to sit for 4 min MAX
Add 7ml of 10% media to the flask to stop the reaction
Add 1ml of fresh media to 4 wells (including the scaffold well)
Transfer the cells = 3ml. 2ml, 1ml, 1ml (see diagram) 3 2 1 1

5. 3ml fresh media per plate
C2C12
3T3
Aspirate
Aspirate
Aspirate
2ml Trypsin, Remove
2ml Trypsin, Remove
2ml Trypsin, Remove
1ml Trypsin, sit 4 min MAX
1ml Trypsin, sit 4 min MAX
1ml Trypsin, sit 4 min MAX
5ml 10% media
7ml 10% media
7ml 10% media
1ml fresh media per well
1ml fresh media per well
Scaffold 3 2 1 1
3ml fresh media per plate 1
0.5 1
3ml
2ml
1ml 2