Differential uptake of large and small anti-PECAM/SA conjugates by HUVECs.

(A-F) Conjugates were prepared at a molar ratio of anti-PECAM to rhodamine-SA of 2:3 (effective diameter, 180 nm; panels A,C,E) and 2:1 (effective diameter, 5130 nm; panels B,D,F). The conjugates were incubated with HUVECs for 1 hour at 37°C, washed, fixed, and counterstained with FITC-conjugated goat antimouse IgG without permeabilization. The rhodamine channel of fluorescence (C,D) was subjected to a threshold function to eliminate pixels with intensity values lower than 128 to generate an image map of conjugates in the field (A,B). The logical “AND” operation between the conjugate image map (A,B) and the image from the FITC-labeled goat antimouse IgG channel (C,D) were used to generate a colocalization map of noninternalized (yellow) particles (E,F). The solid arrows highlight a noninternalized yellow particle, whereas open arrows indicate internalized conjugates (visible only in the red conjugate image map). Bar, 10 μm. (G) Internalization of small (effective diameter, 180 nm) or large (effective diameter, 5130 nm) conjugates by HUVECs was calculated with the use of the number of noninternalized particles (eg, panels E,F) and total conjugates (eg, panels A,B) as processed above. HUVECs showed roughly 3-fold more internalization of small conjugates than of large conjugates.