Editing 293F Protein Production (section)

== Transfect with PEI&nbsp;MAX <br>  ==

'''PEI''': (Linear: Polysciences cat # 23966 or PEI Max: 24765-2). Dissolve in 25mM HEPES and 150 mM NaCl (pH 7.5), 1 mg/ml. Sterile filtered using a 0.22 μM syringe filter, aliquoted, and frozen at -20°C.<br> 

<br>'''Valproic acid (VPA) '''(Sigma cat. No. P4543-100G). 250 mM VPA&nbsp;sodium salt in water, filter through 0.22 μM. Store at -20 oC.<br> 

<br>DNA to PEI ratio: 1:3<br> 

Amount: <br> 

DNA: 3 μg/ml<br>PEI: 9 μg/ml<br><br> 

1.&nbsp; 24 hours prior to transfection, split cells to a density of ~1 x 10^6 cells/ml, in 50 ml medium, and culture overnight in the CO2 incubator with shaking at 37°C. Cell density should be ~2 x 10^6 at the time of transfection. 

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2. Count cells using a hemocytometer.<br> 

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3. Spin down (1000 rpm, 2 min) a sufficient volume to resuspend the cells at a density of 2.5 x 10^6 cells/ml in fresh 293F Freestyle Media in the desired volume for transfection.<br> 

Transfections should be performed at a cell density of between 2.5 x 10^6 and 3.0 x 10^6 cells/ml.<br><br> 

For example:<br> 

culture density of the suspension culture stock = 2.8 x 10^6 cells/ml.<br>For a final transfection volume of 100 ml,&nbsp; 50 ml of cells at a density of 2.5 x 10^6 cells/ml is needed.<br>V1 = 2.8 x 10^6 cells/ml = 50 ml X 2.5 x10^6 cells/ml<br>V1 = 44.6 ml<br>Spin 44.6 ml at 1000 rpm for 2 min and remove media from cell pellet.<br>Resuspend cells in 50 ml of fresh pre-warmed Freestyle 293 medium.<br>Return the culture to the CO2 incubator while preparing DNA and PEI mixtures for transfection. Reserve an aliquot of the culture for cell counting to confirm cell density.<br> 

<br>4. For 50 ml transfection:<br>Add 150 μg DNA to ~ 250 μl of pre-warmed DMEM medium (final DNA conc is 0.5 μg/μl)<br> 

Dilute 1mg/ml PEI&nbsp; to 0.5 μg/μl in DMEM. Final amount is 450 μg PEI. So add 450 μl&nbsp; 1mg/ml PEI to 450 μl DMEM.<br> 

5. Mix DNA with PEI solution, incubate for 20 min. <br> 

6. Add to cells. Return cells to incubator, .<br><br>7. After 24 hr, dilute the cells 1:1 with (''i.e.'', 50 ml) pre-warmed Freestyle 293 Medium supplemented with final conc of 2.5 mM VPA (add 500 μl of 250 mM VP stock solution to 50 ml pre-warmed Freestyle 293 Medium, and add this medium to culture). (For 293E cells, optimal conc is 3.75 to 4 mM of VPA).<br> 

8. Harvest the transfected cells 3-6 days after transfection. Protein production can be monitored by taking small samples (~100 μl) at 24 hr intervals, clarifying the media by centrifugation and analysis by SDS-PAGE&nbsp; to determine the optimal time for harvest.<br><br> 

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