ES Cell Culture for Blastocyst Injection.doc

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Replacing page with 'Image:escellculture.tiff')
Current revision (17:39, 29 June 2009) (view source)
 
Line 1: Line 1:
-
[[Image:escellculture.tiff]]
+
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.

Current revision

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.

Personal tools