Editing 293E Protein Production (section)

== 293E Cell culture<br>  ==

All reagents added to cultures and media must be sterile. 

'''Calf serum, cosmic '''(HyClone) 

Thaw the frozen serum thoroughly at 37°C. Heat-inactivate for 30 min in a 56°C bath, swirling the bottle occasionally for thorough heat distribution. Store aliquots at -20°C. 

'''Cell lines''', 293E or 293-6E (NRC-BRI) 

Other HEK293 cell lines (e.g., 293T, 293S, and 293F) can also be used, but with somewhat lower r-protein yields. 

Use appropriate culture medium listed below, depending on cells used. 

Culture medium, prewarmed, for 293E cells (e.g., '''LC-SFM medium''' [custom low-calcium HSFM formulation; Invitrogen]) 

Supplement with '''1% (v/v) serum and 0.1% (w/v) Pluronic F-68 '''prior to use. 

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Culture medium, prewarmed, for 293-6E cells (e.g., F17 culture medium [Invitrogen] or HyQ SFM4Transfx-293 [HyClone]) 

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The best media for 293E expression is:<br> 

1:1 mixture of Free Style and SFM4Transfx-293 plus 100 ug/ml G418<br> 

'''Free Style'''<br> 

Cat #: 12338026, 12338-026, 6X1 L, $519<br> 

http://www.lifetechnologies.com/order/catalog/product/12338018<br> 

SFM4Transfx-293<br>Cat #: SH30860.LS, 6 x 1L<br>http://www.thermoscientific.com/en/product/hyclone-sfm4transfx-293-media.html<br><br><br> 

Add '''10''' mL of Pluronic F-68 stock solution per liter of culture medium to a final concentration of 0.1% (w/v) prior to use. 

DMSO, cell culture grade (Sigma) 

G418, 50 mg/mL stock solution (Invitrogen) 

caution PBS containing 25 mg/mL '''Erythrosin B''' (Sigma) 

'''Pluronic F-68''', '''10%''' stock solution (w/v) (Invitrogen)

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