The GFCC′C" face of the CEACAM1 N-terminal domain contributes to homophilic adhesion.

The 96-well Immulon 3 plates were coated with goat antihuman Fc prior to the addition of the mutated CEACAM1-3-Fc proteins or of the unmodified CEACAM1-3-Fc and CEACAM1-4-Fc soluble constructs. CD14-Fc was used as the negative control. CHO-CEACAM1-4L transfectants were labeled with the fluorescent tag, BCECF-am, and allowed to adhere to these soluble constructs for 60 minutes at 37°C. The total fluorescence of each well was then determined using the Cytofluor II fluorescence plate reader. The plates were then washed and the number of cells adhering determined by fluorescence estimations in the Cytofluor II as a percentage of the total cells added per well. CHO-Neo cells were used as a negative control for CHO cell binding. Six replicates were used for each construct and the experiments performed on 3 separate occasions. The results show the mean ± SD of 6 replicates for one typical CHO-Neo experiment and for 3 CHO-CEACAM1-4L binding experiments.