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Fig. 2. The donor-acceptor model facilitates quantification of nascent N-cadherin-dependent intercellular adhesions. (Ai-ii) N-cadherin immunostaining of donor cell (asterisk) cultured on incompletely established acceptor monolayer shows minimal staining of N-cadherin at intercellular junctions of cells in acceptor layer and enriched band of N-cadherin staining at donor-acceptor interface (open arrows) but not at donor-substratum interface (closed arrow) after a 15-minute incubation. Bar, 20 µm. (Bi-v) Flow cytometry shows fluorescence intensity and spectral separation of the following samples in donor:acceptor model: (i) bare:bare; (ii) bare:RITC-dextran; (iii) FITC-dextran:bare; (iv) FITC-dextran:RITC dextran. (v) Cytospins of sorts from regions R5 and R6 of flow cytograph (iv) demonstrate the accuracy of flow cytometry analysis and show no detectable crossover of fluorescence between detection channels. (Ci) When donor cells are harvested with trypsin and EGTA (R2 C EGTA), temporal increases of intercellular adhesion seen in control samples (R2 C) harvested with trypsin and calcium do not occur. Data are representative of three independent experiments and show means±s.e.m. (ii) Maintaining calcium in trypsinization medium for donor cells preserves the integrity of the extracellular domain of surface-expressed N-cadherins. Positive control of cells permeabilized with paraformaldehyde fixation are included. (D) Cells treated with GC-4, an N-cadherin specific blocking antibody, and an HAV domain mimetic peptide show 60% (P<0.05) and nearly complete blockade (P<0.01), respectively, of intercellular adhesion. Cells treated with 4B4, a ß1-integrin blocking antibody and a corresponding scrambled HAV mimetic peptide show intercellular adhesion ratios similar to standard growth conditions.

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