Brefeldin A problem

[Ian A. York]

	I'd appreciate suggestions from people who have worked with 
brefeldin A.  We want to block glycoprotein transport in a T cell line.  
To test the function of the BFA, we activated the cells, in the presence 
of BFA, and measured the surface appearance of activation markers.  The 
one we have looked at most is produced de novo on activation, so if the 
BFA is working there should be no apparent upregulation.  

	To our surprise, there was a significant population that showed 
levels as bright as in the absence of BFA.  We then tried increasing 
levels of BFA (from 5 to 50 ug/ml) (5 ug/ml is our usual concentration).  
We were even more surprised.

	At the lowest concentration, there was a clear biphasic 
population - that is, either expression was blocked altogether (about 
half the cells), or it was expressed normally (the other half).  For a 
couple of higher concentration points, the proportion of negatives 
increased, to a maximum of about 70%; again, the remainder showed normal 
intensity.  Then, as the BFA concentration increased more, the cells 
shift back to normal intensity.  At 20 ug/ml, over 75% of the cells are 
of normal intensity.  At 50 ug/ml, the population is no longer biphasic.  

	We also tried pretreating the cells, though only at a low 
concentration of BFA; it made no difference compared to treating 
simulataneously with the activation.

	I'm puzzled.  Anyone got any suggestions?

Ian
-- 
Ian York   (york at mbcrr.harvard.edu)
Dana-Farber Cancer Institute, 44 Binney St., Boston MA 02115
Phone (617)-632-4328     Fax  (617)-632-2627