The base medium for this cell line is Prigrow X series medium available at abm, Cat. No. TM4074. Carbon dioxide (CO2): 5%, Temperature: 37.0
Applications
For Research Use Only
Subculture Protocol
1. Subculture when the culture reaches 90% confluency or above. 2. Prepare Poly-L-Lysine coated culture flask (2 mug/cm square) one day before subculture. 3. Warm complete medium, TrypsinEDTA solution, TNS, and DPBS (Ca++ and Mg++ free) to room temperature. We do not recommend warming reagents and medium in a 37°C water bath prior to use. 4. Rinse the cells with DPBS. 5. Add 8 ml of DPBS and then 2 ml of TrypsinEDTA solution into flask. Gently rock the flask to ensure complete coverage of cells by T/E solution. Incubate the flask in a 37°C incubator for 1 to 2 minutes or until cells completely round up. Use a microscope to monitor the change in cell morphology. 6. During incubation, prepare a 50 ml conical centrifuge tube with 5 ml of FBS. 7. Transfer T/E solution from the flask to the 50 ml centrifuge tube (a small percent of cells may detach) and continue to incubate the flask at 37°C for another 1 to 2 minutes (no solution in the flask at this moment). 8. At the end of incubation, gently tap the side of the flask to dislodge cells from the surface. Check under a microscope to make sure that all cells detach. 9. Add 5 ml of TNS to the flask and transfer detached cells to the 50 ml centrifuge tube. Rinse the flask with another 5 ml of TNS to collect the residual cells. 10. Examine the flask under a microscope for a successful cell harvest by looking at the number of cells being left behind? there should be less than 5%. 11. Centrifuge the 50 ml centrifuge tube at 1000 rpm for 5 minutes. Resuspend cells in culture medium. 12. Count and plate cells in a new Poly-L-Lysine coated culture vessel.