Hoechst 33258 fluorescent dye

Cultured cells were washed in ice cold PBS, fixed (1% PFA, 20 min, RT), permeabilized (100% methanol, 15 min on ice) and stained with the following antibodies: anti-p44/42 MAPK (ERK1/2; 137F5; Cell Signaling); secondary antibody Goat anti-Rabbit IgG (A-11,012; Alexa Fluor 594; Thermo Fisher) at times and concentrations recommended by the manufacturer. For cell nuclei visualization, Hoechst 33258 fluorescent dye (Sigma-Aldrich) was used 5 min before measurement. Analysis of the samples was performed using the Amnis® ImageStream® Mk II imaging cytometer (Luminex Corporation, Austin, Tx, USA). The data obtained were then analyzed using Ideas software (Luminex Corporation) and its built-in module for the analysis of nuclear translocation. Briefly – after gating the cells based on their size and aspect ratio (singlets determination) and focus gradient, the shape of each cell and its nucleus was determined by the built-in algorithm. The correlation of Hoechst 33258 (nucleus) and AF 594 (ERK1/2) signal intensity within the cell was evaluated, and the Similarity Median (SM) parameter was calculated. The area containing cells with signs of translocation was gated using the SM parameter, and cells within this gate were scored as „ERK translocated“. Representative images of the cells, dot plots, and gates are shown in Supplementary Fig. S1.

Cultured cells were harvested and washed in PBS/0.5% BSA and incubated for 30 min on ice with Alexa Fluor 700 labeled anti-CD45 monoclonal antibody (mAb) (clone 30-F11; BioLegend), APC labeled anti-CD11b mAb (M1/70; BioLegend), FITC labeled anti-H-2Kb/H-2Db mAb (MHCI; 28-8-6, BioLegend), FITC labeled anti-I-A / I-E mAb (MHCII; M5/114.15.2, BioLegend), FITC labeled anti-CD80 mAb (16-10A1; BioLegend), PE labeled anti-CD86 mAb (PO3; BioLegend), PE/Cy7 labeled anti-CD301b mAb (URA-1; BioLegend) or PE labeled polyclonal Ab anti-TNFSF14 (LIGHT; Bioss Antibodies, Woburn, MA, USA). Unstained cells were used as controls. A total of 50 000 cells were analyzed after exclusion of dead cells and debris. Five minutes before measurement, Hoechst 33258 fluorescent dye (Sigma-Aldrich) was added and used to exclude dead cells. Data were collected using the LSR II cytometer (BD Bioscience, Franklin Lakes, NJ, USA) and analyzed using GateLogic 400.2 A software (Invai, Mentone, Australia). Representative dot plots and histograms illustrating the gating strategy are shown in Supplementary Fig. S3.

The freeze-dried powder was dissolved in distilled water to prepare a 0.5% suspension. After conventional smear preparation, the composition of the slurry was qualitatively analyzed by toluidine blue (Sigma-Aldrich, St. Louis, MO, USA) and collagen type II (Sigma-Aldrich, St. Louis, MO, USA) staining. Hoechst 33258 fluorescent dye (Sigma-Aldrich, St. Louis, MO, USA) was used to detect the cell residues. The sizes of the fibers in the slurry were observed by scanning electron microscopy (SEM; Olympus BX51, Japan). The scaffold was fixed in 10% neutral formalin for 12 h, dehydrated with gradient alcohol, embedded in paraffin, and sectioned into 5 μm-thick sections. These were used for histological and immunohistochemical staining.