Cloning CTL's in New Species - How?

Mitch Magee MMAGEE at TCID.TDH.STATE.TX.US
Mon Apr 8 07:42:21 EST 1996


>     So, how would I go about cloning and propagating antigen-specific
> CTL's?  In the bad old days before Il-2, did people use irradiated feeder
> cells?  Would PBMC feeders suffice?  Sacrificing an animal and isolating
> splenocytes would be out of the question for my experiment.  I want to
> accomplish this with bleeding only.

You could take peripheral blood and stimulate it with Concanavalin A 
(I used 5 micrograms Con A/ml with a cell concentration of 5 x 
10^6/ml).  After 48 hours, spin the culture to remove cells, add 
alpha-methyl mannoside to bind residual Con A, and filter through a 
0.22 micron filter.  Use this conditioned media at a 10% final 
concentration with your cultured cells.  I have had very good luck 
with this source of growth factors.  



Mitch Magee
Department of Clinical Investigation
Texas Center for Infectious Disease
2303 S.E. Military Dr.
San Antonio, TX  78223-3597

Phone:  (210) 534-8857 xt 2209
Fax:    (210) 532-7791
Email:  MMAGEE at tcid.tdh.state.tx.us




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