Establishment and maintenance of EBV-transformed B-cell lines

5-10x106 freshly separated PBMCs are incubated for 1 hour in 1ml of supernatant from an EBV-producing marmoset cell line B958.

The cells are then diluted with 9ml of RPMI/15% FCS containing 1ug/ml Cyclosporin (CsA) and put into a small T/C flask.

Usually colonies of transformed B-cells (BCL or B-LCL) grow out in the form of large clumps of cells within two weeks. Sometimes the process takes 4-6 weeks.

The BCL are maintained in culture in R10 [RPMI/10% FCS/1%Glutamine/1%P+S], and split once or twice weekly depending on the rate of growth. They should be regularly tested for mycoplasma, and stocks of mycoplasma-free cells frozen down in liquid nitrogen.

B958 cell line

Cells are seeded into small T/C flasks using R10 at a concentration of 0.5x106cells/ml.

After 7-10 days the supernatant is harvested. The cell suspension is centrifuged at 3000rpm/15 minutes, and 1ml aliquots of supernatant are stored at +4C.