View source for Freezing C2C12 and Satellite Cells

==Freezing C2C12 and Satellite cells==
===Methods===
* Mr. Freeze box with isopentane
* Cryotube (in tissue culture box) 
* Warm PBS, basic FGF
*Trypsin
* Gelatin coated dish (cold room, satellite cells only)
*Basic FGF recipe (keep for three days in a 50 ml tube) >>> Use 2.7 ul of basic FGF for satellite cells, the rest is prolif. Media

===Freezing C2C12 cells===
# Remove old media 
# 5 ml of PBS to wash for 10 seconds and remove
# 1 ml of trypsin for 5 minutes until cells detach  
# Pipette 3ml of prolif media into 15 ml tubes (one for each plate of cells being frozen) 
# Once cells are detached add the 1 ml of cells into the 3ml of prolif media 
# Centrifuge the 15 ml tubes (in this example three 15 ml tubes) to collect cells at the bottom [centrifuge at 1.3g for 5 minutes] 

'''Prepare cryotube media'''
* 900 ul of prolif media  
* 100 ul of DMSO (cabinet) 
# Mix solution three times each tube
# After centrifuge, aspirate the pellet (remove the liquid) tip the 15 ml tube to vacuum out the liquid with a pipette tip, its okay if a little media is left over. 
# Resuspend cells by tapping or dragging the tube 
# Take up the media from the cryotube and place into the 15 ml tube of cells, flush a little, take up everything from the tube and place back into the cryotube
# Seal and place cryotubes into the Mr. Freeze box. Close the box and put in the -80 freezer for at least 24 hours

===Freezing Satellite cells: ???===
# Remove old media
# 1 ml of trypsin (no PBS wash)
# Pipette 3ml of basic FGF media in each tube and 8 ml into the gelatin coated dish
# Flush 5-6 times on dish
# Take the full volume into the 15 ml tube and pulse in pipette
# 100 ul goes in the dish (spread the five dots) rest goes in a 15 ml tube 
# Flush petri dish, label (date, name, cell type, passage)
# Shake dish gently in incubator