|Name of Cells: |
|Human Liver Stellate Clls(CI-HLiStCs) |
CI-HLiStCs (UBP-0071) are selected from HLiStCs (UBP-0035) infected with lentiviruses expressing SV40 LT antigen under the control of Tet-On system resistant puromycin. CI-HLiStCs are maintained with proliferative capacity in the presence of DoxycyclineNOTE1 in Stellate-Growth medium (contains 10% serum and growth supplements, Cat# UBP-31).
Characterization of the cells Cytoplasmic alpha-SMA: >99% positive by immunofluorescence
Cytoplasmic PECAM1 < 1% positive by immunofluorescence
CI-HLiStCs are negative for HIV-1, HBV, HCV, and mycoplasma.
Product Use: CI-HLiStCs are for research use onlyNOTE3.
Shipping: Frozen Vial.
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°CNOTE2 water bath, and then transfer the cells into a T25 flask pre-coated with Universal Coating Solution (UBP01) as described in detail in Subculture Protocol.
- 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.
- Rinse the cells in T25 flask with 5ml HBSS (Room Temperature, RT) twice.
- Add 2ml of Universal Detachment Solution (RT) (UBP-23) into one T25 flask (make sure the whole surface of the T25 flask is covered with Universal Detachment Solution), and gently dispose the excessive Universal Detachment Solution within 60 seconds with aspiration.
- 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.
- Add 5ml Universal Neutralization Buffer and spin the cells down with 800g for 5 minutes.
- Re-suspend the cell pellet with 10 – 20ml of SGM full medium and the cell suspension is transferred directly into 2 or 4 pre-coated T25 flasks (5ml each, and the cells are sub-cultured at 1:2 to 1: 4 ratios)
- Change medium every 2-3 days and cells usually become confluent within 7 days (when split at a 1:4 ratio).
NOTE 1: To minimize the effect of SV40 antigen LT for your studies, cells could be cultured in the absence Doxycycline for 3-5 days.
NOTE 2: Although wild type SV40 LT antigen is used for cell immortalization, we noticed that the immortalized cells are growing better at 33-34ºC, we encourage the end users to switch to 33-34ºC if cells are growing slower at 37ºC.
NOTE 3: The purchase of this product conveys to the buyer the nontransferable right to use the purchased amount of the product and all replicates and derivatives for research purposes conducted by the buyer in his/her laboratory only (whether the buyer is an academic or for-profit entity). The buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes.