Figure 5.

A_2AR activation decreases cell surface targeting of NPC1 in mouse IPMФs. (A) Immunofluorescence staining of mouse IPMФ cells was made using NPC1 specific, primary and Alexa-488 conjugated anti-rabbit secondary antibody (green). The nuclei of macrophages were stained with DAPI (blue). NPC1 specific fluorescence intensity was measured after LPS activation and treatment with the A_2AR agonist CGS21680 by High Content Analysis, as described in the Section 2. 66–145 fields and 300–3470 cells were acquired per well, and laser-based autofocus was performed at each imaging position. Images of DAPI and Alexa-488 channels were collected at 2 μm of the Z image plane using a 63× water immersion objective (NA: 1.15). Cellular features, such as the number of spots and relative spot intensities in the (B) membrane and (C) cytoplasmic regions, were extracted. Data obtained from the individual analysis of 300–3470 different cells are presented as mean ± SEM. # p < 0.05 LPS vs. LPS + A_2AR agonist treated cells.