Hi, I would like to know about the mouse myeloma cell line P3X63Ag8.653 azaguanine resistancy period upon subculture. My cells have undergone 9 passages in total and then I decided to cryopreserve it. Upon revival last month they show 70% viability. My question is,even though the cells has been sensitized with 8 azaguanineprior to buying , do i still need to sensitized it because the cells has undergone few passages? I’m also wondering if myeloma cells can undergo infinite passages and will the cells loose the sensitization to azaguanine along the way? What would be the maximum passage for the myeloma cells because i will need to revive the cells again for fusion and it will undergo few passages again.

Answer

P3X63Ag8.653 is a subclone P3X63.Ag8 which was originally selected based with 8-aza-guanine sensitivity. Periodic growth in 8-asa-guanine ensures cells are HGPRT negative and thymidine kinase negative. Growth on 6-Thio-Guanine is useful for periodic selection for HAT sensitivity. Also remember that cells for fusion should not be passaged for a long times so keep frozen stocks and do not maintain the cells for more than several passages prior to fusion. It is also good practice to dilute the myeloma cells to about 200,000 cells/ml the day before fusion. Finally, I think your viability should be greater than 70% after you thaw the cells. Try bringing a cup of 37C water to your frozen cells and thawing as quickly as possible at 37C until the cells are completely thawed.