To analyse the cytoskeleton of the macrophages, the actin cytoskeleton was visualized. Macrophages were harvested after 5 days culture, reseeded on glass coverslips and then activated with IFNγ/LPS or IL-4 to induce M1 or M2 subsets. M0 macrophages were cultured in medium without stimuli. After 48 h, cells were left untreated or activated with CCL2 10 μM (Peprotech, Heerhugowaard, The Netherlands) for 10 minutes fixed with 4% paraformaldehyde in PBS for 30 minutes at 4°C, rinsed with PBS and permeabilized with 0.2% Triton X-100 in PBS for 5 minutes. The cells were rinsed with 10 mM Glycine, rinsed with PBS, and incubated with Cdc42 (1:500 Abcam, Cambridge, UK) for 1 h. The cells were rinsed with PBS prior to staining with the second antibody goat anti rabbit ALEXA 488 (1:400, Invitrogen) and rhodamine phalloidin (1:300, Sigma Aldrich) a high affinity, fluorescent filamentous actin probe for 1 h [9 (link)]. Cells were washed twice and the nuclei counterstained using Hoechst (1:5000, Sigma Aldrich) and washed again and mounted in mounting medium (88% hydrolysed polyvinyl alcohol, Dako, Heverlee, Belgium). Images were captured on a Leica DM6000 microscope.
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