TransIT-PRO® Use in the Expi293™ Expression System

Protocol for Transfecting Expi293F™ Cells using TransIT-PRO® Reagent

1. Determine the total number of cells required for your experiment at least one day prior to transfection. Each 30 ml transfection requires 7.5 × 10⁷ cells in 25.5 ml of Expi293™ Expression Medium.
2. Seed the cells at a density of 2.0 × 10⁶ viable cells/ml and maintain 18 – 24 hours prior to transfection to ensure that cells are actively dividing at the time of transfection. Shake flasks overnight on an orbital shaker (125 rpm when using a shaker with a 2 cm orbital throw) at appropriate temperature and CO₂ levels (e.g. 37°C, 8% CO₂).
3. On the day of transfection, quantitate cell number and viability using an automated cell counter or using the trypan blue dye exclusion method. Do not proceed with transfection unless cells are >95% viable.
4. Determine the volume of cell suspension containing the number of cells needed for one transfection. Each 30ml transfection requires 7.5 × 10⁷ cells (i.e. 2.5 × 10⁶ cells/ml final concentration).
5. Add the appropriate volume of cell suspension to each sterile, disposable 125 ml Erlenmeyer shake flask and bring up the volume to 27 ml by adding fresh, pre-warmed Expi293™ Expression Medium for each 30 ml transfection. Return the cells to the incubator.
6. For each 30 ml transfection, prepare reagent-DNA complexes as follows:
   • In a sterile tube, dilute 30 μg of plasmid DNA in Opti-MEM® I Reduced Serum Medium (Life Technologies) to a total volume of 3 ml. Mix gently but thoroughly.
   • Add 30 μl of TransIT-PRO® Transfection Reagent to the tube containing diluted DNA. Mix gently but thoroughly.
   • Incubate the DNA and TransIT-PRO® Transfection Reagent mixture for 20–30 minutes at room temperature to allow transfection complexes to form.
   NOTE: This procedure recommends a 1:1 TransIT-PRO®:DNA ratio, which results in the optimal titers for most proteins. However, customer feedback indicates that a 2:2 ratio (i.e. 60 µl TransIT-PRO® + 60 µg DNA per 30 ml culture volume) may result in improved protein expression with some constructs.
7. After the DNA-TransIT-PRO® Transfection Reagent complex incubation is complete, add 3 ml of DNA-TransIT-PRO® Transfection Reagent complex alone to each shaker flask from Step 5. To the negative control flask, add 3 ml of Opti-MEM® I medium instead of DNA-TransIT-PRO® Transfection Reagent complex. Each flask should contain a total volume of 30 ml.
   NOTE: No enhancer addition is required with the TransIT-PRO® Reagent.
8. Shake flasks on an orbital shaker (125 rpm when using a shaker with a 2 cm orbital throw) at appropriate temperature and CO₂ levels (e.g. 37°C, 8% CO₂).
9. Cells or media (if recombinant protein is secreted) may be harvested beginning at approximately 48 hours post-transfection and assayed for recombinant protein expression. The incubation time for optimal protein expression depends on the nature of the recombinant protein.

Expi293™ and Expi293F™ are trademarks of Life Technologies Corporation.