CI-HNCs (UBP-0070) are selected from HNCs (UBP-0036) infected with lentiviruses expressing SV40 LT antigen under the control of Tet-On system resistant puromycin. CI-HNCs are maintained with proliferative capacity in the presence of Doxycycline in HNCs Growth Medium (contains 10% serum and growth supplements, UBP-34).

The cells are positive for several neuronal markers:

Handling of Arriving Cells: 

When you receive the cells in a frozen vial, you can transfer the vial of cells into a -80ºC freezer for short-period storage or a liquid nitrogen tank for long-term storage.  Thaw the cells in a 37°C water bath, and then quickly transfer the cells into a T75 flask with 15 ml MSCGM in the presence of 1ug/ml of Doxycycline and incubated overnight in a 37ºC^, CO2 incubator and change the medium next day (15 ml complete MSCGM) and every other thereafter.

Subculture Protocol

1. Pre-coating of T25 flasks: Add 2ml of Universal Coating Solution (UBP-01) into one T25 flask and make sure whole surface of the flask is covered with the coating solution. Five minutes later, dispose excessive Universal Coating Solution by aspiration and the flask is ready to be used (no need for overnight incubation when using Universal Coating Solution).
2. Rinse the cells in T25 flask with 5ml HBSS (Room Temperature, RT) twice.
3. Add 2ml of Trypsin/EDTA (RT) (UBP-23) into one T25 flask (make sure the whole surface of the T25 flask is covered with Universal Detaching Solution and gently dispose the excessive Trypsin/EDTA solution within 60 seconds with aspiration.
4. Leave the T25 flask with the cells at 37C for extra 1-2 minute (the cells usually will detach from the surface within 1-2 minutes). You can monitor the cells under microscope and when most of cells become rounded up, hit the flask against the bench surface, and the cells will move on the surface of the flask when monitoring under microscope.
5. Add 5ml Universal Neutralization Buffer and spin the cells down with 800g for 5 minutes.
6. Re-suspend the cell pellet with 10 – 15ml of HNCs Growth Medium and the cell suspension is transferred directly into 2 or 3 pre-coated T25 flasks (5ml each, and the cells are sub-cultured at 1 : 2 to 1 : 3 ratios)
7. Change medium every 2-3 days and cells usually become confluent within 7 days (when split at a 1:4 ratio).