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

Figure 8.

Figure 8

A2AR activation decreases EEA1 expression in mouse IPMФs. (A) Immunofluorescence staining of IPMФ cells was made using EEA1 specific (SAB4300682 Sigma-Aldrich, St. Louis, MO, USA) primary and Alexa-488 conjugated anti-rabbit secondary antibody (A27034, Thermo Fisher Scientific, Waltham, MA, USA) (green). Nuclei of macrophages were stained with DAPI (blue) (D1306, Thermo Fisher Scientific, Waltham, MA, USA). EEA1 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). Fifty fields and 370–3445 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 370–3445 different cells are presented as mean ± SEM. ## p < 0.01 LPS vs. LPS + A2AR agonist-treated cells.