| A new system which allows precise control of the initiation, the duration, and the area of contact between a T cell and an antigen-presenting cell, has been developed. This system consists of a well with four drains and two glass cover slips. One cover slip is fixed at the base of the well which is then filled with liquid medium and the other cover slip is mobile, supported by surface tension of air-liquid interface in the well. T cells which are loaded with a fluorescent dye, and antigen presenting cells are adhering at the base and the top cover slip, respectively. The distance between two cover slips is controlled by lowering or raising the air-liquid interface. At a defined time zero, by lowering the top cover slip onto base cover slip, antigen presenting cells are brought into contact with T cells and initiated the cell-cell contact. After a desired time period, medium is re-introduced into the well, lifting the top cover slip and the cell-cell contact is broken. The contact area between T cells and antigen presenting cells are controlled with fiducial microbeads. The bead fixes the distance between two cover slips to its diameter and thus controls the degree of T cell-antigen presenting cell contact. Using the system, the intracellular calcium concentration of T cells is monitored over time at the single cell level, to quantitatively study the activation of T cells. |
