Fitc labelled collagen

For live-cell imaging, cells were seeded (100,000 cells per quadrant) on Cellview culture dishes (Greiner bio-one, 627870) which were coated with 10 μg/ml FITC-labelled collagen (Sigma, C4361) in PBS for 1 h at room temperature in the dark. Cells were stimulated with LPS (100 ng/ml) for 24 h.
After collagen uptake in a 24 well plate (coated as described above) macrophages were seeded on glass coverslips in RPMI for 30 min to attach and fixed using 4% paraformaldehyde (PFA, 15 min at 4 °C) followed by four PBS washes. Cells were blocked and permeabilised for 30 min at 4 °C with CLSM buffer (PBS + 20 mM glycine + 3% BSA) and 0.1% saponin followed by an overnight staining with the primary antibody (Cathepsin B, Calbiochem IM27L and LAMP1, Biolegend 328601) 1:200 diluted in CLSM with saponin at 4 °C. The next day cells were washed twice with PBS +0.1% saponin and incubated for 30 min at room temperature in CLSM +0.1% saponin with secondary antibodies donkey-anti-mouse IgG (H&L) labelled with Alexa 647 and donkey-anti-rabbit with Alexa 568. After washing the cells with PBS with 0.1% saponin, the coverslips were mounted on glass slides in 67% glycerol containing Trolox (1 mM) and DAPI (0.33 μg/ml). Samples were imaged using an LSM800 Zeiss microscope with a 63 × oil immersion lens.

A 24-wells plate was coated with 10 μg/ml FITC-labelled collagen (Sigma, C4361) in PBS for 1 h at room temperature in the dark. After incubation, wells were twice washed with PBS. Next, 100,000 macrophages were seeded into the wells and cultured for 24 h with LPS, 4-OI, or both (adding 4-OI 1 min prior to LPS). The next day, the samples were washed with PBS and macrophages were detached using StemPro Accutase (Fisher Scientific, 11599686) for 10 min in the cell culture incubator followed by a e780 fixable live/dead staining (1:1,000, 15 min at 4 °C), fixation in 4% PFA, and analysis by flow cytometry. The addition of inhibitors SB203580 (10 μM) and ML385 (10 μM) in DMSO was done prior to the addition of cells and supplementation with 4-OI or LPS.