Immortalization of B lymphocytes by EBV is an effective procedure for inducing long-term growth of certain human B lymphocytes. The basic protocol described below to accomplish this can be divided into three stages: preparation of virus, preparation of target cells to be immortalized, and EBV infection and growth of infected cells.
CAUTION: EBV may cause disease in nonimmune individuals. Only individuals with serum antibodies to EBV should handle cell lines infected with the virus. In addition, EBV has been associated with a number of lymphoproliferative disorders. Biosafety practices must be followed (see Chapter 7 introduction)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique must be used accordingly.
Materials
Complete RPMI-10 medium (APPENDIX 2), without and with 1 μg/ml cyclosporin A
(written request from Sandoz; prepare 1 mg/ml stock in 100% ethanol and store
at −20°C)
B95-8 cells (marmoset cell line; ATCC #CRL 1612)
Heparinized peripheral blood (APPENDIX 3)
Phosphate-buffered saline (PBS; APPENDIX 2)
Hanks balanced salt solution (HBSS; APPENDIX 2)
Beckman centrifuge with JS 4.2 rotor (or equivalent)
0.45-μm filter
50-ml conical tubes
25-cm2 tissue culture flasks
Additional reagents and equipment for cell counting, cryopreservation, and
determination of viability (APPENDIX 3) and Ficoll-Hypaque gradient
centrifugation (UNIT 7.1)
