EVB-transformed B cell transfection

[Kevin Mulcahy]

I wish to transfect tumour-associated antigens into human 
EBV-transformed B cell lines. However, I have come across two main 
problems.

Firstly, I understand that EBV-transformed B cell lines are notoriously 
difficult to transfect. Therefore, does anybody use a transfection 
method which has provided them with "satisfactory" results with 
EBV-transformed B cell lines?

Secondly, if transfectants are obtained, I need a procedure to clone 
them. However, using normal growth medium (RPMI/10% FCS), I have had no 
success with serial-dilution cloning or soft-agar cloning using normal 
growth medium. From the literature, I gather that the use of human 
endothelial cell supernatant (HECS) greatly increases the ability of 
EBV-transformed B cell lines to grow when there is only one or two cells 
per well. Does anybody know if HECS can obtained commercially or do I 
have to prepare it in-house?

Any advice is greatly appreciated.
Best regards,

Kevin Mulcahy
(e-mail: K.Mulcahy at sheffield.ac.uk)