Formation of stable conjugates with target cells. (A) OT-I CD8⁺ T cells (labeled in red with the fluorescent lipophilic dye PKH26) were mixed at 37°C in the presence or absence of 25 μM latrunculin B with EL4 target cells (labeled in green with CFSE) that had either been pulsed (+pOVA) or not (−pOVA) with OVA257-264 peptide. After 1 hour, cells were fixed and analyzed by flow cytometry. The percentages of T cells found conjugated to target cells indicated in the dot plots were calculated as follows: 100 × [(PKH26⁺ CFSE⁺ events)/(total number of PKH26⁺ events)]. (B) Percentages of conjugates formed by OT-I cells with their targets in the presence of the indicated inhibitors, calculated as in panel A. Gray represents the “background” level of conjugates formed with target cells in the absence of antigen, and black represents the level of conjugates formed with target cells in the presence of antigen. Levels of statistical significance were calculated using the Student t test; ***P < .001. (C) As in panel A except that MD4 B cells were stained with the B cell–specific B220 mAb before mixing them with CFSE-labeled J558L or J558LmHEL cells. The percentages of B cells found in conjugates with target cells were calculated using the following formula: 100 × [(B220⁺ CFSE⁺ events)/(total number of B220⁺ events)]. (D) As in panel B but for MD4 B cells. Bars represent standard deviation; n = 4.