Fac scan platform

In the mouse model, we followed the protocol of a previous study [27 (link)]. Wild-type and TG2-null mice fully backcrossed onto BL57/6 background [31 (link)] were injected intraperitoneally with 1 ml PBS containing 3 mg MSU crystals (prepared as described earlier). During the final preparations the MSU crystals were washed in PBS. Injection of PBS alone had no effect on the cytokine formation or the neutrophil cell number injected into wild-type mice. Mice were sacrificed at the indicated time points and peritoneal cells were obtained by lavage with 2 ml sterile PBS. Total cell numbers were counted and percentage of neutrophil cells was determined by labeling the cells with fluorescein isothiocyanate (FITC)-conjugated anti-GR1 (BD Biosciences) antibody. Samples were analyzed on Becton Dickinson FACScan platform. Active TGF-β1 and IL-1β were determined from centrifuged, cell-free peritoneal lavage fluid by ELISA (eBioscience and R&D Systems, respectively). Mice were maintained in specific-pathogen-free condition in the Central Animal Facility and all animal experiments were approved by the Animal Care and Use Committee of University of Debrecen (DEMÁB).

Cells were collected after they were treated with different concentrations (0–100 μg/mL) of FAC in the presence or absence of PDTC for the indicated times. The cell surfaces were blocked with 15% sheep serum at 4°C for 15 min and were then washed twice with phosphate buffer solution (PBS, pH 7.2). Then, cells were incubated with monoclonal antibodies against CD40, CD80, CD86, and the corresponding fluorescent markers for 30 min at 4°C. After cells were washed twice with PBS and resuspended in PBS, they were subjected to flow cytometry with a FAC Scan platform (Becton Dickinson).

RAW264.7 cells (1 × 10⁵ cells/mL) were seeded in 6-well plates and treated with various concentrations (0–10 μg/mL) of FAC in the presence or absence of PDTC for 24 h. Then, cells were collected, washed once with cold PBS, fixed in 75% cold alcohol overnight at 4°C, and washed twice with cold PBS. Fixed cells were resuspended in 100 μL RNase at 37°C and incubated with 400 μL propidium iodide (PI) at 4°C in a dark room for 30 min. Cell cycle progression was analyzed by using flow cytometry with a FAC Scan platform (Becton Dickinson).