Trouble obtaining T cell clones!

Mon Oct 2 14:15:18 EST 1995

Dear colleagues,

I have been having limited success obtaining murine T cell clones 
against my antigen of interest. I easily get T cell lines 
isolated from the inguinal nodes of Balb/c mice immunised 
subcutaneously with antigen emulsified in complete Freund's adjuvant. 
However it is proving difficult to establish T cell clones from these 
lines (passaged every 14 days in the presence of antigen and IL-
2) even though their proliferative responses to antigen are quite 

I clone the cells in 96 well plates in the presence of 1 million 
mitomycin-C treated spleen cells, 50 ug/ml antigen and 10 U/ml IL-2. 
I have varied the numbers of T cells/well from 10 to 500, however, I 
either get no clones or too many (suggesting that the positive wells 
are not monoclonal). 

As there are not many detailed T cell cloning protocol around, I'd 
appreciate any comments, suggestions or tips that anyone has. Many 

Cloneless in Montreal


J. Darren Campbell
Institute of Parasitology,
Macdonald Campus of McGill University
21,111 Lakeshore Road, Ste-Anne-de-Bellevue, 
Quebec, Canada H9X 3V9

e mail: Darren at
Tel: 514-398-7997
fax: 514-398-7857

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