Stem Cell culturing

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

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

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

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