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. 2023 Jun 7;45(6):4948–4969. doi: 10.3390/cimb45060315

Figure 6.

Figure 6

A2AR activation decreases cell surface targeting of LAMP2 in RAW 264.7 cells. (A) Immunofluorescence staining of RAW 264.7 cells was made using LAMP2 specific, Alexa-488 conjugated antibody (green). The nuclei of macrophages were stained with DAPI (blue). LAMP2-specific fluorescence intensity was measured after LPS activation and treatment with the A2AR agonist CGS21680 by Opera Phenix High Content Confocal System (Perkin Elmer, Waltham, MA, USA). 154–166 fields and 550–5450 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 550–5450 different cells are presented as mean ± SEM. * p < 0.05 control (vehicle-treated) vs. LPS activated cells; # p < 0.05 LPS vs. LPS + A2AR agonist-treated cells.