Phalloidin conjugated to alexa fluor 633

Frozen peripheral blood mononuclear cells (PBMCs) from healthy donors and patients were thawed and CD14⁺ cells selected using magnetic beads (Miltenyi). 2 x 10⁵ cells/ well in a 24 well plate were seeded on 10ug/ml fibronectin-coated cover slips (R&D systems) in 500ul 20ng/ml macrophage colony stimulating factor (MCSF, Gibco) for 6 days to obtain monocyte-derived macrophages (MDMs). Cells were fixed with paraformaldehyde 4% (Thermo Fisher Scientific) for 10 minutes on ice followed by 8% for 20 minutes at room temperature, permeabilised with 0.1% triton (Sigma) for 5 minutes at room temperature and non-specific binding reduced by blocking with 5% BSA/PBS for 1 hour at room temperature. Cells were incubated with primary anti-vinculin antibody (Sigma 1:200) for 1 hour at room temperature, washed twice with PBS and incubated with secondary antibody conjugated to Alexa Fluor 488 (1:500 Life Technologies) and phalloidin-conjugated to Alexa Fluor 633 (1:200 Thermo Fisher Scientific) for one hour at room temperature. Cells were washed twice with PBS and cover slips mounted onto slides using mounting solution with DAPI for nuclear staining (ProLong Diamond Antifade Mountant with DAPI, Life Technologies) overnight. Slides were imaged using Zeiss 710 confocal microscope at 63x magnification and podosome analysis was carried out on at least 100 cells per sample from 10 fields of view.