Fig 1: AGE-2 and AGE-3 effects on scavenger receptor expression. (a) RAW264.7 cells seeded at 2 × 105 per well incubated for 1 h with 200 µg/ml AGE-2, AGE-3, or BSA were stained with specific FITC-labelled Abs against CD163 or CD206, specific PE-labelled CD204 or LOX-1, and APC-labelled CD36 or RAGE, and relative mean fluorescence intensities (MFIs) determined by flow cytometry (n = 6). Each column represents respective surface protein MFIs relative to medium only (Untreated, Unt) after 1 h incubation, arbitrarily defined as 100%. Data are presented as the means ± SEM and analysed using one-way ANOVA followed by Tukey’s test. ***p < 0.001, **p < 0.01 compared with the Unt value. (b) For RAW264.7 cells seeded at 2 × 105 per well incubated with increasing concentrations AGE-2, AGE-3, and BSA (0.2–200 µg/ml) for 1 or 4 h, relative CD204 MFIs were determined by flow cytometry (n = 4–6). Each column represents MFIs relative to Unt after 1 or 4 h incubation, arbitrarily defined as 100%. Data are presented as the means ± SEM and analysed using one-way ANOVA followed by Tukey’s test. ***p < 0.001, *p < 0.05 compared with Unt. Insert in 1 h shows overlay of representative monoparametric histogram of CD204 expression in RAW264.7 cells incubated with medium only (Untreated; greyscale), 200 µg/ml BSA (orange), AGE-2 (blue), or AGE-3 (red line). (c) RAW264.7 cells seeded at 4 × 105 per well were cultured for 4 h with 200 µg/ml Alexa Fluor 488 (green)-labelled BSA, AGE-2, or AGE-3. CD204 intracellular localisation was visualized using anti-CD204 Ab and Alexa Fluor 594 (red) labelled secondary Ab, with confocal fluorescence microscopy. Rabbit IgG served as an anti-CD204 Ab isotype-matched control. All micrographs are shown at the same magnification (×400). Scale bar indicates 50 µm. Each BSA, AGE-2, or AGE-3 panel indicates, as follows; (upper) Alexa Fluor 488-labelled AGEs, (middle) Alexa Fluor 594-labelled CD204, or (lower) merged image. Clipped images at the upper left of the merged panel in AGE-2 or AGE-3 represent higher magnifications of the white rectangle region in the corresponding panel. Scale bar indicates 10 µm.