F actin alexa fluor 488 or alexa fluor 568 phalloidin

MØP were differentiated by M-CSF for 6 d and stimulated with LPS for 24 h as described above. Subsequently, cells were fixed with 3.7% formaldehyde, permeabilized with 0.5% Triton X-100 and PBS (pH 7.5) for 10 min and stained for podosome markers F-actin (Alexa-Fluor-488– or Alexa-Fluor-568–phalloidin; Molecular Probes, Eugene, OR) and vinculin (mAb V9264, 1:500; Sigma-Aldrich) to highlight podosome rings. CellMask Deep Red plasma membrane stain (no. C10046; Thermo Fisher Scientific) was used to stain the cytoplasm and nuclei at a concentration of 2 μg/ml. Images of fixed samples were acquired with a confocal laser-scanning microscope (DMI 6000 with a TCS SP5 AOBS confocal point scanner; Leica) equipped with an oil-immersion HCX PL APO 63× numerical aperture 1.4–0.6 objective. Podosome number and cell area analysis were performed using an ImageJ (National Institutes of Health, Bethesda, MD) macro (38 (link)). The analysis was performed with three different donors with 30 cells for each condition (n = 3 × 30).