A. Three-color fluorescence microscopic image of PFA fixed
LSEC from 2BKIX mice, which where incubated with HIV-488 plus HIVIG-594 immune
complex (IC) at 37°C/4°C for 30 min. (a, f) Green puncta identify
HIV particles. (b, g) Red puncta identifies HIVIG. (c, h) DAPI (blue) identifies
nucleus. (d, i) DIC (e, j) Merged (a)–(d)/(f)–(i). (a-e) Confocal
microscopy images showing endocytosis of LSEC at 37°C, (f-j) confocal
microscope images showing binding of HIV-488 plus HIVIG-594 IC by LSEC at
4°C. Scale bar indicates 5μm. B. Live cell confocal
microscopic image of LSEC from 2B-KIX mice incubated with HIV plus HIVIG-FITC IC
for various time points from 3 to 30 min at 37°C (top panel) and
4°C (bottom panel). Green puncta identify HIVIG FITC. C. Bar
graph showing quantification of green puncta of HIV plus HIVIG-FITC IC from 25
live cell confocal microscopic images with and without trypan blue is shown
here. The mean fluorescence intensity and the total pixel area of green puncta
were measured and plotted. A total of 400 LSEC from three different
mice/biological replicate were analyzed. Bar graphs show average fluorescence
intensity and mean± SD per cell. Values of all significant correlations
(p<0.05) are given with degree of significance indicated (*
p<0.05, ** p<0.01, *** p<0.001) by students t-test.