Controlling duration of contact between T cells and antigen-presenting cells.
A new method which allows precise control of the duration of contact between T cells and antigen-presenting cells (APCs) has been developed. A glass coverslip coated with poly-L-lysine, and then with T cells, was placed at the base of a cylindrical well, and the well was filled with liquid medium. A round coverslip, on which APCs were adhered, was supported on the surface of the medium by surface tension, cell-side down. By withdrawing medium from four capillary holes near the base of the well, the coverslip could be lowered to initiate contact between T cells and APCs at a defined time zero. The contact was broken at desired time points by re-introducing medium into the well in order to separate the two coverslips. Each cell type remained adherent to its original surface after separation for all contact times studied. The T cells were monitored for intracellular calcium mobilization using the fluorescent dye, Fura-2. Contact durations of less than 1 min did not trigger calcium signals. Contact durations of 3 and 5 min induced strong calcium signals. Breaking the contact caused a rapid decrease in intracellular calcium levels. This method of cell manipulation allows precise control of the duration of contact of T cells with APCs, while keeping the cells under continuous observation. The measurements so obtained provide a quantitative understanding of the dynamics of early T cell activation.
Kim
S
S
Department of Chemical Engineering and Applied Chemistry, Columbia University, 500 W. 120th Street, New York, NY 10027, USA.
Braunstein
N S
NS
Leonard
E F
EF
Thomas
J L
JL
D000818:Animals; D017951:Antigen Presentation; D002450:Cell Communication / Q000276:immunology; D008213:Lymphocyte Activation; D051379:Mice; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology*