Rhodamine phalloidin

J774 macrophages were seeded overnight in DMEM containing 10% FBS and 1% penicillin/streptomycin, on round 15 mm cover glass. J774 were pre-incubated with rytvela (1 μM), for 30 min and incubated with IL-1β (100 ng/ml) for 4 h. Then medium was removed and Fluorescein-labeled Escherichia coli K-12 BioParticles mix was added with HBSS. Two hours later, cells were washed twice with PBS for 5 min, fixed in 4% PFA for 30 min, and permeabilized in 1.0% Triton X100 and blocked in 10% FBS 1 h. Cells were counterstained with Rhodamine Phalloidin (1:500, 1 h) (Santa Cruz Biotechnology, R415) and DAPI (1:5000; 5 min) (Sigma–Aldrich, D9542) to evidence cell-contour and cell-nuclei. Phagocytosis efficiency was assessed using a confocal microscope (Zeiss, LSM 510).

Cells on fibers were fixed in 4% paraformaldehyde, permeabilized in 0.1% Triton X100 solution and blocked in 5% goat serum. Paxillin (Tyr31) staining was done using primary rabbit anti-Paxillin antibodies (Invitrogen) at a dilution of 1:250 and incubated at 4 °C for 1 h. Secondary goat anti-rabbit Alexa Fluor 488 (Invitrogen) antibodies were incubated for 45 min at room temperature in the dark. For double immunostaining, total Paxillin (Host: Mouse, Invitrogen) primary antibodies at a dilution of 1:40 and Phospho-FAK (Tyr397) (Host: Rabbit, Invitrogen) primary antibodies at a dilution of 1:200 were used and incubated for 4 h. Secondary antibodies goat anti-mouse Alexa Fluor 647 and goat anti-rabbit Alexa Fluor 488 were used at dilution of 1:150 and incubated for 1.5 h at room temperature. F-Actin stress fibers were stained using Rhodamine Phalloidin (SantaCruz Biotechnologies). Cell nuclei were stained with 300 nM of DAPI (Invitrogen) for 5 min. The scaffolds were kept in 2 ml antifade imaging solution during imaging. Fluorescent images were taken using an Axio Observer Z1 microscope (Carl Zeiss). Actin live cell imaging was performed as per the manufacturer’s instructions on using the reagent CellLight Actin-RFP, Bacman 2.0 (Invitrogen).