Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

Hui-Yuen, Joyce; McAllister, Shane; Koganti, Siva; Hill, Erik; Bhaduri-McIntosh, Sumita

1. Visualization of cells by light microscopy: By a week after EBV infection, clusters of cells are visible by light microscopy. Figure 3 shows an example of early microscopic clusters ( Figure 3B ) in a flask. 2. As time progresses, microscopic clusters become larger such that clumps are visible macroscopically in the flask. Figure 3C shows larger clusters of cells in established LCL by light microscopy. 3. Feeding cells: Double the volume of culture medium in the culture flask on day 12. Subsequently, expand culture by increasing its volume 2-3 fold using complete RPMI. 4. Periodicity of feeding cells should be determined based on rate of cell growth. When the culture medium turns yellow, it is generally time to replace the media as above. Typically, this occurs once per week. However, some cell lines may need to be fed more or less frequently. Expand culture to 75-100 ml over the next few weeks. 5. Cryopreservation: When freezing down cells, centrifuge cells at 600 x g for 10 minutes at room temperature. Remove the supernatant and resuspend cell pellet in cold freezing medium containing 90% FBS and 10% dimethylsulfoxide at 1 x 10 7 cells/ml. Place tube in liquid nitrogen. . Un-infected cells ( Figure 2A ) and cells exposed to EBV from HH514-16 cells (Figure 2B ), harboring a non-immortalizing strain of EBV, do not demonstrate CD23 hi CD58 + cells.