F. BMDM cells were treated with 0.004% DMSO or 100 nM MLi2 prior to live cell surface staining with Alexa-594 conjugated rat anti-mouse CD11b antibody on ice for ten minutes. Three wells of cells from each condition were fixed and imaged for initial surface CD11b binding (shown in red as indicated). Six wells of cells from each condition were further incubated at 37°C to allow rapid internalization of CD11b-antibody complexes for ten minutes prior to striping of surface CD11b antibody using 0.5% acetic acid on ice for one min. Three of the six wells were fixed and surface-stained with Alexa-488 conjugated goat anti-rat antibody to show CD11b-antibody complexes remaining on the cell surface (shown in green as indicated). Internalized CD11b-antibody complexes are shown in red. The other three wells of cells were further incubated at 37°C again for ten min to allow recycling of internalized CD11b-antibody complex followed by surface staining with Alexa-488 conjugated goat anti-rat antibody to show CD11b-antibody complexes recycled back to the surface. Representative images from n=3 biologically independent experiments are shown. Scale bars represent 30 μm. G-I. (G) The initial surface CD11b, or (H) internalized CD11b, or (I) recycled CD11b were calculated from >20 images analyzed for each condition from n=3 biologically independent experiments. Each dot represents the mean value from one experiment. Error bars show ± SEM with significance assessed by Mann-Whitney test. * represents P<0.05. 