Isolated human islets were dispersed into single cells using Accutase (Millipore), ~1500-2000 islet equivalents\/ml. 20,000-65,000 islet cells were transferred to individual wells of a 96 well culture plate (CELLSTAR, Greiner Bio-one) for immediate staining for flow cytometry analysis or for culture prior to staining. For intracellular staining, isolated islet cells were fixed in 2% paraformaldehyde (Sigma-Aldrich) and permeabilized using 0.3% saponin (Sigma-Aldrich). The cells were stained with 10E4 mouse anti-human HS mAb (10E4, 1\/50; US Biological\/Amsbio), mouse anti-mouse Col18 mAb (1\/50; Santa Cruz Biotechnol.) or the corresponding isotype control Ig (mouse IgM\u03ba or IgG2b\u03ba; BD Biosciences) followed by goat anti-mouse Ig-R-PE (1\/100; Southern Biotech). The geometric mean fluorescence ratio (GMFR) was calculated by dividing the geometric mean fluorescence intensity (GMFI) of cells stained with primary mAb by the GMFI obtained with the relevant isotype control Ig. Cells were analyzed using a BD LSRI flow cytometer and CellQuest\u2122 Pro software (version 6.0; BD Biosciences).