ES Cell Culture for Blastocyst Injection.doc

ES Cell Culture for Blastocyst Injection:

Media: Filter sterilize all media.

LIF Media (250mL; 10%FBS) 250 mL GMEM 2.5 mL Amino Acids 2.5 mL L-glutamine 2.5 mL Sodium Pyruvate 0.28 mL 2-mercaptoethanol (1000x stock) 25 mL FBS 0.5 mL LIF

10% FBS Media w/o LIF (25mL) 25 mL GMEM 0.25 mL Amino Acids 0.25 mL L-glutamine 0.25 mL Sodium Pyruvate 0.028 mL 2-mercaptoethanol (1000x stock) 2.5 mL FBS

Blast Injection Media 10 mL GMEM 1.0 mL FBS 0.275 mL 1M HEPES

Cell Culture:

D0: Thaw a vial of cells. Resuspend in 10mL LIF Media Pellet Wash with 5mL LIF Media Pellet Resuspend in 10mL LIF Media and plate onto a gelatinized T25

D1: Feed cells with 10mL LIF Media or split (1:10) if needed D2: Feed cells with 10mL LIF Media or split (1:5) if needed

D3: Feed cells with fresh LIF Media as early as possible 10:00-10:30 Trypsinize cells off of T25 Resuspend in 9 mL 10% FBS Media w/o LIF Split 1:5 into a new T25 Pellet 4mL of cells. Resuspend in 2mL Blast Injection Media and Count Dilute cells to 1x106 cells/mL with Blast Injection Media *I provide Paul with 1-2 mL of cells at 1x106 cells/mL. *He also needs the excess Blast Injection Media.