The interaction of chitosan with monocytes, granulocytes, and lymphocytes in whole blood. (a) Whole blood was incubated for 30 minutes with 5 μg/ml rhodamine B isothiocyanate (RITC)-80% deacetylated (80 M) chitosan at 37°C for 30 minutes before analysis by flow cytometry. The binding index was calculated as fluorescence units of each leukocyte population incubated with RITC-80 M chitosan/fluorescence units of leukocyte population in the absence of RITC-chitosan. Results are presented as mean ± standard error. P values from Student's two-tailed unpaired t-test: **P < 0.005 versus autofluorescence for each leukocyte population. (b) Macrophages were seeded on glass slides (2 × 106 cells/ml) in RPMI 1640 supplemented with 0.1% decomplemented fetal bovine serum, pre-stained with 1 μg/ml calcein AM for 30 minutes at 37°C, and incubated with 100 μg/1 × 106 cells RITC-zymosan for 1.5 hours (a positive control), 15 μg/ml RITC-80 M, or RITC-95% deacetylated (95 M) chitosan for 3 hours at 37°C. Macrophages were then visualized live through a spinning disc confocal microscope with a 63× objective. The top panels are images taken in the X-Y plane and the lower panels are images taken in the X-Z plane. This figure represents the results of three independent experiments.