Flp-In™-Jurkat Cell Line

Flp-In Jurkat cells are a little tricky to handle and are very sensitive to centrifugation. You may try the following protocol to thaw the cells:

- Thaw 1 vial of cells into a T25 flask containing 5-7 mL of fresh medium (without selection). Do not spin down the cells to remove the DMSO at this point, as they are very sensitive to handling (including centrifugation). Typically, there is a lot of debris present upon thaw.
- 24 hours post-thaw, spin down the cells at 900 rpm for 2-3 minutes and resuspend gently into 5 mL of fresh medium. Spinning down the entire volume of cells greatly reduces the amount of debris in the culture.
- At day 5 or 6, it is okay to add the selection. Continue to passage the cells (spinning the cells down) every 2-3 days for a total of 1.5 weeks, each time resuspending back into only 5-7 ml of fresh medium in a T25 flask to build up the cell density. The Jurkat cells are very small and clumpy, and they expand very slowly.
- Attempt to expand the cells into a T75 flask only after about 1.5 weeks.

In theory, one can get multiple integrations of the Flp-In expression construct—an FRT-specific integration event and a random, second-site integration. However, random integration is a relatively uncommon event. Limiting the amount of DNA in the transfection will reduce the chance of second-site integration. We have transfected 293 cells (lacking the FRT site) with the pcDNA5/FRT vector and have identified one potential second-site integrant after screening over 200 clones. DNA integrations can be detected by Southern blot. A single integrant will display a single band; double: two; triple: three, etc. We have maintained a number of Flp-In expression cell lines for over four months and have not observed any loss of the Flp-In expression construct, whether hygromycin selection was maintained or not.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

We offer the Flp-In T-REx system that contains pFRT/lacZeo and pcDNA6/TR stably integrated into HEK 293 cells. This cell line has been functionally tested for its ability to regulate expression.