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| ES Cell Culture for Blastocyst Injection:
| | [[Image:escellculture.tiff]] |
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| Media: Filter sterilize all media.
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| '''LIF Media (250mL; 10%FBS)'''
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| 250 mL GMEM
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| 2.5 mL Amino Acids
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| 2.5 mL L-glutamine
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| 2.5 mL Sodium Pyruvate
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| 0.28 mL 2-mercaptoethanol (1000x stock)
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| 25 mL FBS
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| 0.5 mL LIF
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| '''10% FBS Media w/o LIF (25mL)'''
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| 25 mL GMEM
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| 0.25 mL Amino Acids
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| 0.25 mL L-glutamine
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| 0.25 mL Sodium Pyruvate
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| 0.028 mL 2-mercaptoethanol (1000x stock)
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| 2.5 mL FBS
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| '''Blast Injection Media'''
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| 10 mL GMEM
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| 1.0 mL FBS
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| 0.275 mL 1M HEPES
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| '''Cell Culture:
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| '''
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| D0: Thaw a vial of cells.
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| Resuspend in 10mL LIF Media
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| Pellet
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| Wash with 5mL LIF Media
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| Pellet
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| Resuspend in 10mL LIF Media and plate onto a gelatinized T25
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| D1: Feed cells with 10mL LIF Media or split (1:10) if needed
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| D2: Feed cells with 10mL LIF Media or split (1:5) if needed
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| D3: Feed cells with fresh LIF Media as early as possible
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| 10:00-10:30 Trypsinize cells off of T25
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| Resuspend in 9 mL '''10% FBS Media w/o LIF'''
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| Split 1:5 into a new T25
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| Pellet 4mL of cells. Resuspend in 2mL Blast Injection Media and Count
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| Dilute cells to 1x106 cells/mL with Blast Injection Media
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| I provide Paul with 1-2 mL of cells at 1x106 cells/mL.
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| He also needs the excess Blast Injection Media.
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