40 



freezing vials (Vanguard International) in 0.5ml aliquots 

 (210ijg/vial) . The labeled Con A was thawed one time only 

 and the preparation was used within 18 days of its synthesis, 



It should be noted that successful labeling was acheived 



125 

 only with I batches used within five days after their 



arrival . 



Cell binding measurement . Methods for the determina- 



125 

 tion of I-Con A binding to cultured CHO H-7, H-7Wcr and 



rat A-9, LAN-2 cells were generally adapted from those pre- 

 sented by Noonan and Burger (1973a) . Cells were grown to 

 approximately 80% confluency in Linbro multiwell plates with 

 30mm diameter wells (Linbro Scientific). For binding, cul- 

 tures were removed to room temperature for 5 minutes, media 

 were removed by aspiration washed gently two times with 

 0.15M PBS, pH 7.4 and 1.0ml of the desired concentration of 



I-Con A (2x 10 cpm/yg) in PBS was added to duplicate 

 cultures. After 15 minutes incubation the Con A was re- 

 moved followed by 5 washes of 2.0ml PBS. PBS containing 

 0.05M EDTA (l.Oml/well) was added and the cells were 

 allowed to remain at room temperature for 30 minutes. Tryp- 

 sin-EDTA (0.5ml) was added to each well and the plate incu- 

 bated at 37°C for 15 minutes. A final addition of 1.0ml 

 20% Triton X-100 was made and the plates heated at 40°C 

 for 60 minutes. Duplicate 0.5ml aliquots were removed from 

 each well to 10ml of Brey's solution and the activity deter- 

 mined by counting in a Beckman LS-133 liquid scintillation 

 counter set on the narrow tritium window. Each experiment 



