Ly6c pe cy7

Mouse leukocytes were isolated from the BM as previously described (16 (link)). Briefly, legs were excised from mice after either sham, ischaemia only or IR injury surgeries. BM was collected from femurs and tibias into expansion media (DMEM, high glucose supplemented with 8.9% FBS, 1% MEM-D-valine, 0.1% sodium bicarbonate, 1% penicillin/streptomycin, 1% MEM non-essential amino acids, 1% MEM vitamin mix, 10 U/ml interferon-γ pH 7.4). Erythrocytes were lysed using ACK, the suspension washed twice with expansion media and resuspended into PBS containing 0.1% BSA. Cells were then incubated for 1 h at 4°C with primary antibodies towards IL-36R, IL-36α/β and appropriate IgG controls. Cells were washed with PBS containing 0.1% BSA before incubating with AF647-labelled donkey anti-goat. Leukocyte sub-types were identified using fluorescently labelled antibodies towards neutrophils (PE anti-mouse Ly-6G/GR-1; Biolegend) and monocyte/macrophages (PE-Cy7 Ly6C; Biolegend). Antibodies identifying leukocyte populations were split into separate samples to avoid spectral overlap and then fixed with 2% formyl saline. 50,000 events were collected on a Beckmann Coulter CyanADP with compensation performed on the Summit 4.4.00 software. Data was analysed using FlowJo v10.7.2.

To identify binding capability of each aptamer on the K562 and Molm14 cell lines, cells were resuspended in 2% FBS and stained with DAPI at a 1:10,000 dilution before they were applied for flow cytometry analysis. To assess aptamer binding on PBMCs, cells were first incubated with fluorescently labeled antibodies, and then stained with DAPI before flow cytometry analysis on an LSRII Flow Cytometer or a BD FACS Fortessa (Bronte et al., 2016 (link)). Antibodies used for flow cytometry analysis included FITC-CD45 (BD Biosciences, United States), APC-Cy7- CD45 (BD Biosciences, United States), PE-CD11b (Tonbo, United States), AF700-Ly6G (Biolegend, United States), FITC-Ly6G (Biolegend, United States), PE-Cy7-Ly6C (Biolegend, United States). Results were analyzed using the Flowjo software.

Murine IL‐6 cytokine was purchased from R&D Systems. Murine GM‐CSF cytokine GM‐CSF was purchased from Abcam. Anti‐Ly6G (1A8) was purchased from eBioscience. STAT3 inhibitor was purchased from BioVision. Ethanol was from Sigma–Aldrich. Anti‐phosphorylated STAT3 (p‐STAT3) was purchased from CST. APC‐CD11b, FITC‐Ly6G, and PE‐Cy7‐Ly6C antibodies were purchased from BioLegend. APC‐CD3, FITC‐CD4, and APC‐Cy7‐CD8 antibodies were purchased from BD Biosciences. Berberine hydrochloride was purchased from Sigma–Aldrich. Lieber‐DeCarli liquid diet was purchased from Bio‐Serv.