Cell aggregation mediated by subtype-specific homophilic interaction in trans of plexins B2 and B3. Aggregation of NIH-3T3 cells expressing full-length recombinant B3, L1, or B2 and non-transfected cells (100 × magnification). (a) Negative control non-transfected cells labeled with DiO (green) were mixed 1:1 with DiI-labeled ones (red) and incubated in DMEM for 45 min prior to fluorescence microscopy. The formed aggregates contain approximately equal proportions of DiO- and DiI-labeled cells. (b)-(d) Non-transfected cells labeled with DiO and DiI-labeled cells stably expressing full-length B3 (b), L1 (c), or B2 (d) were mixed 1:1, respectively and treated as described in a. Predominance of DiI-labeled cells in the formed aggregates indicates cell-cell-adhesion due to homophilic interaction in trans mediated by B3, L1, and B2. (e) L1-transfected cells labeled with DiI and DiO-labeled cells stably expressing B3 were mixed 1:1. The almost pure red or pure green aggregates indicate preferential homotypic interactions of both B3 and L1, respectively. (f-h) As additional negative control cells labeled with DiO and expressing B3 (f), L1 (g) or B2 (h) were mixed 1:1 with DiO labeled cells expressing B3 (f), L1 (g) or B2 (h). The formed aggregates contain approximately equal proportions of DiO- and DiI-labeled cells.