Figure 2.

T cell activation as measured by intracellular calcium elevation. Jurkat T cells were loaded with calcium-sensitive dye, Fluo-4 AM, whose fluorescence intensity increases with intracellular concentration of Ca²⁺. (a) Normalized fluorescence intensities of T cells stimulated by particles with the reverse (total number of cells: 96) and the native “bull’s eye” pattern (total number of cells: 111) are plotted on a color scale and sorted based upon the fluorescence intensity of the first peak. (b, c) Calcium responses of two representative cells that are in direct contact with the “bull’s eye” patterns are plotted as a function of stimulation time. Time zero is defined as the time when cells are flown into imaging chambers. White arrows indicate Janus particles that are in contact with cells. Anti-CD3 and fibronectin are shown in red and green, respectively. Scale bars: 5 µm. (d) Calcium response of T cells that are in contact with the “bull’s eye” patterns is quantified with three parameters: the fraction of activated T cells (% activation), activation intensity (average fluorescence amplitude) and duration of calcium response (response fraction). Average fluorescence amplitude and response fraction are plotted in scattered plot with the mean (± SEM, shown in red). For the native pattern, a total of 30 cells were analyzed, and for the reverse pattern, 24 cells. Either data set represents results from more than 10 experiments done over 4 different days.