A. Thawing and culturing cells

1. The culturing should be done as soon as possible upon receipt of cultures.

2.  Remove the vial from dry ice or liquid nitrogen, and immediately incubate it in a water bath at 37^oC.

3.  When ice has melted (should only take less than a minute), remove the vial from water bath and wipe tube with 70% ethanol.

4.  Perform all manipulations after step 4 under strictly aseptic condition, in a sterile room or hood.

5.  Transfer the content of the vial into a flask contaiing at least 10 volumes of culture medium.

6.  Place the flask in the incubator set at recommended temperature and aeration.

Note

1.If the cells can not be thawed and cultured immediately upon receipt, the vial should be held at -70^oC or below, preferably in the vapor phase of liquid nitrogen.

2.It is recommended that the culture medium be replaced with fresh medium 24 hours after thawing to remove the protective freezing agent.

3.If it is desirable to remove the freezing agent immediately, centrifuge the diluted suspension at 100Xg for 10 min, and then resuspend the pellent in an appropriate volume of culture medium.