An apparatus which allows precise control of the time of initiation and the
area of contact of cells with immobilized ligands has been developed. Cell
s are trapped in an asymmetric film that can be quantitatively thinned, for
cing the cells into close contact with ligands adsorbed on the base of the
apparatus. Using microbeads to indicate the film height, the amount of thin
ning can be controlled to within 1 mu m, producing known contact areas betw
een cells and the ligand-coated surface. This system was used with anti-CD3
-coated surfaces of different densities to examine the effect of ligand den
sity on T cell activation, while keeping the number of ligands presented to
the cells constant. T cell activation was observed individually in each ce
ll as intracellular calcium mobilization. In these experiments both the per
cent of T cell activation and the rate of calcium rise were found to depend
only on the number of anti-CD3 molecules presented and not on their densit
y. This implies that the spacing between molecules is not important in the
range studied, and suggests that receptor clustering to levels higher than
dimers may not be necessary for induction of calcium mobilization by anti-C
D3. (C) 2000 Elsevier Science B.V. All rights reserved.