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Frequently Asked Questions/Troubleshooting
1. What cell line were the VE-cell lines derived from?
The VE-cell lines are engineered from Sf9 cells that were originally derived from the ovarian tissue of the fall armyworm (Spodoptera frugiperda).
2. Do I need to keep the VE-cell lines under neomycin selection to prevent loss of expression of the vankyrin protein?
No, the VE-cell lines are stably transformed cell lines and neomycin is not needed to maintain vankyrin protein expression.
3. What is the average size of the VE-cells?
While derived from Sf9 cells the VE-cells are larger in size than the parental Sf9 cells. VE-cells are approximately 20 microns in diameter.
4. What media may I grow my VE-cells in?
The VE-cells are grown in Sf900 II media (Invitrogen™). However, the cells may be adapted to Sf900 II media supplemented with 10% FBS or in Expression Systems’ ESF 921 media.
5. How many passages may I maintain my cells?
We recommend starting new cells after they have been through 30 passages. A passage is considered anytime you add media to dilute your cells.
6. How do I start my VE-cell lines?
Thawing Cells Thaw frozen cells rapidly in a 37°C water bath. Decontaminate the outside of the vial with 70% ethanol before transferring the cell suspension either into a T-25 cm² flask (adherent culture) or a 125 mL Erlenmeyer flask (suspension culture).
Adherent Culture: Put 0.5 mL of thawed cells into 5 mL of media and transfer flask to a 27°C incubator and allow the cells to attach for 30-45 minutes before replacing the medium with 5 mL fresh Sf-900 II SFM. Change the medium after 24 h. Subculture cells when they have reached a density of >80% confluency. Cells should be split at a 2:5 dilution to maintain log cell growth.
Suspension Culture: Put 1 mL of thawed cells in 12 mL of media and incubate Erlenmeyer flask in a 27°C incubator on an orbital shaker platform (1-inch orbit) rotating at 100-110 rpm. Loosen caps of flasks to allow proper oxygenation/aeration. Once a cell density of >2x106 viable cells/mL has been reached (approx. 7-10d), cells should be diluted to a density of 6x105 cells with Sf-900 II SFM.
How do I freeze my cells down?
Freeze cells at a density of >2x107 viable cells/mL in a freezing medium composed of fresh Sf-900 II serum free medium (Invitrogen™), 10% heat-inactivated FBS and DMSO to a final concentration of 7.5%. (Optional freezing media: 50% conditioned Sf-900 II media: 50% fresh Sf-900 II and DMSO to a final concentration of 7.5%). Centrifuge cells at 100g at 4°C for 5-10 minutes, remove the supernatant and re-suspend the pellet in an appropriate volume of chilled freezing medium to reach a density of >2x107 viable cells/mL. Transfer suspension into a cryovial. Place cells in a styrofoam container and place at -20°C for one hour, then transfer the styrofoam container with cells to –80°C overnight before transferring the cells to liquid nitrogen (vapor phase). Frozen cells remain viable if properly stored in liquid nitrogen.
Which VE-cell line should I purchase?
10010 (previously VE-CL-01) displays the most prolonged enhanced protein expression (up to 7 days post-infection) when using conventional BEVS viruses. We recommend 10010 for laboratories working with highly stable intracellular and secreted proteins as the pronounced enhancement of viability in these cells allows for prolonged expression and accumulation of recombinant protein over time.
10020 (previously VE-CL-02) displays a sharp peak of enhanced protein expression at day 3 and day 4 post-infection. We recommend 10020 for laboratories working with highly unstable or toxic proteins as enhanced protein expression occurs at a very high level and over a very short time interval in these cells.
10030 (previously VE-CL-03) displays an intermediate phenotype to 10010 and 10020 showing significantly enhanced protein expression and moderately enhanced longevity. Due to its intermediate expression properties, we recommend 10030 for laboratories working with proteins of unknown toxicity and stability or for general enhancement of most recombinant proteins expressed in conventional recombinant BEVS viruses.