Editing 293EF Protein Production (section)

== Transfection<br>  ==

=== 30 mL Transfection<br>  ===

Final transfection volume: 30 mL<br> 

*Number of cells to transfect: 7.5 × 107 cells (final cell density of 2.5 × 10^6 cells/mL) with &gt;95% viability<br> 
*Amount of plasmid DNA: 30 μg<br> 
*Amount of ExpiFectamine 293 Reagent: 40–100 μL (we typically use 80 μL).<br> 
*Use 2.7 μL ExpiFectamine 293 Reagent per 1 μg of plasmid DNA transfected<br><br>

1. The day before transfection (day 0), determine the number of cells. For each 30-mL transfection, 7.5 × 10^7 cells in 25.5 mL of Expi293 Expression Medium is required.<br> 

<br>2. To transfect cells on the following day (day 1), seed the cells at a density of 2.0 × 10^6 viable cells/mL and incubate at 37°C in a humidified atmosphere of 8% CO2 in air on an orbital shaker rotating at 125 rpm.<br> 

<br>3. On the day of transfection (day 1), determine cell viability using an automated cell counter or the trypan blue dye exclusion method.<br>Important: Cell density should be &gt;3 × 10^6 cells/mL and the viability of cells must be over 95% to proceed with transfection. Cells should not be clumping.<br> 

<br>4. Calculate the volume of cell suspension containing the number of cells needed for one transfection. For each 30 mL transfection, 7.5 × 10^7 cells are required.<br><br> 

5. Add the appropriate volume of cell suspension to each sterile, disposable 125-mL Erlenmeyer shaker flask and bring up the volume to 25.5 mL by<br> adding fresh, pre-warmed Expi293 Expression Medium for each 30 mL transfection.<br> 

<br>6. Incubate the cells in a 37°C incubator with a humidified atmosphere of 8% CO2 in air on an orbital shaker rotating at 125 rpm.<br> 

<br>7. For each transfection sample, prepare lipid-DNA complexes by performing the following:<br> 

a) Dilute 30 μg of plasmid DNA in Opti-MEM I medium to a total volume of 1.5 mL. Mix gently.<br> b) Gently mix the ExpiFectamine 293 Reagent by pipetting it up and down before use.<br> c) Dilute 80 μL of ExpiFectamine 293 Reagent in Opti-MEM I medium to a total volume of 1.5 mL. Mix gently and incubate for 5 minutes at room<br> temperature.<br> Note: Longer incubation times may result in decreased activity.<br> d) After the 5 minute incubation, add the diluted DNA to the diluted ExpiFectamin<span style="background-color: navy; color: white;" />e 293 Reagent to obtain a total volume of 3 mL. Mix gently.<br>e) Incubate DNA-ExpiFectamin<span style="background-color: navy; color: white;" />e 293 Reagent mixture for 20–30 minutes at room temperature to allow the DNA-ExpiFectamin<span style="background-color: navy; color: white;" />e 293 Reagent complexes to form.<br> 

<br>8. After the DNA-ExpiFectamin<span style="background-color: navy; color: white;" />e 293 Reagent complex incubation is complete, add 3 mL of DNA-ExpiFectamin<span style="background-color: navy; color: white;" />e 293 Reagent complex to each shaker flask from Step 6. To the negative control flask, add 3 mL of Opti-MEM I medium instead of the DNA-ExpiFectamin<span style="background-color: navy; color: white;" />e 293 Reagent complex. Each flask should now contain a total volume of 28.5 mL.<br> 

<br>9. Incubate the cells in a 37°C incubator with a humidified atmosphere of 8% CO2 in air on an orbital shaker rotating at 125 rpm.<br><br>10. Approximately 16–18 hours post-transfection, add 150 μL of ExpiFectamin<span style="background-color: navy; color: white;" />e 293 Transfection Enhancer 1 and 1.5 mL of ExpiFectamin<span style="background-color: navy; color: white;" />e 293 Transfection Enhancer 2 to each flask. The final volume should be approximately 30 mL in each 125-mL flask. <br> 

<br>11. Cells or media (if recombinant protein is secreted) may be harvested beginning at approximately 48 hours post-transfection and assayed for<br>recombinant protein expression. Time for optimal protein expression depends on the nature of your recombinant protein.<br><br> 

=== Optimizing Protein Expression <br><br>  ===

Time course experiment: Harvest cells or media at 24, 48, 72, 96 hours post-transfection.<br> 

The Expi293 Expression Medium is designed to support the transiently transfected culture for up to 7 days. Optimal yield of the rabbit IgG<br>clone expressed from the Antibody Expressing Positive Control Vector occurs at 6–7 days post-transfection<br><br> 

=== Scale Up Transfection<br><br>  ===

<br> 

{| cellspacing="1" cellpadding="1" border="1" style="width: 673px; height: 299px;"
|-
| <br> 
| colspan="5" | <br>Transfection Volume
|-
| <br> 
| 1 mL (24-well plate)<br> 
| 2 mL<br>(6-well plate)<br> 
| 30 mL <br> 
| 1 liter <br> 
| 3.8 liter<br>
|-
| Total Number of Cells*<br> 
| 2.5 × 10^6 <br> 
| 5.0 × 10^6 <br> 
| 7.5 × 10^7 <br> 
| 2.5 × 10^9 <br> 
| 9.5 × 10^9<br>
|-
| Amount of DNA<br> 
| 1 μg <br> 
| 2 μg <br> 
| 30 μg <br> 
| 1 mg <br> 
| 3.8 mg<br>
|-
| DNA Dilution Volume (in Opti-MEM I)<br> 
| to 50 μL <br> 
| to 100 μL <br> 
| to 1.5 mL <br> 
| to 50 mL <br> 
| to 190 mL<br>
|-
| Amount of ExpiFectamine 293 Reagent<br> 
| 2.7 μL <br> 
| 5.4 μL <br> 
| 80 μL <br> 
| 2.7 mL <br> 
| 10.3 mL<br>
|-
| ExpiFectamine 293 Reagent<br>Dilution Volume (in Opti-MEM I)<br> 
| to 50 μL <br> 
| to 100 μL <br> 
| to 1.5 mL <br> 
| to 50 mL <br> 
| to 190 mL<br>
|-
| Lipid/DNA Complex Volume<br> 
| 100 μL <br> 
| 200 μL <br> 
| 3 mL <br> 
| 100 mL <br> 
| 380 mL<br>
|}

*Final cell density of 2.5 × 10 cells/mL<br>

Control:<br> 

Transfect 30 mL of suspension Expi293F cells using 30 μL of the Antibody Expressing Positive Control Vector (i.e., 1 μg of positive control per 1 mL of<br> Expi293F culture) following the protocol provided on pages 11–12. The rabbit IgG expressed by the control will be secreted into the Expi293<br>Expression Medium. Expression can be detected by day 3 post-transfection; however maximum yield is obtained 6–7 days post-transfection. The expected<br>yield of the control IgG is approximately 200–300 mg IgG per liter of culture on day 7<br><br>