Ly6g pe clone 1a8

Ubc-GFP donor animals were subjected to the experiments either untreated or after one week of treatment with PBS or colchicine (0.25 mg/kg BW) injected intraperitoneally once daily. Single cell suspensions were prepared from the bone marrow as described above. Neutrophils and monocytes were isolated by staining the cells with Ly6G-PE (clone 1A8) and CD115-biotin (clone AFS98, both BioLegend) followed by linking to magnetic beads (anti-PE and streptavidin microbeads, Miltenyi Biotec, Bergisch Gladbach, Germany) and enrichment on magnetic-activated specific columns (130-042-401, Miltenyi Biotec). The degree of purification of the separated cells was determined by flow cytometry.
Equal cell amounts were injected intravenously into Apoe^-/- mice 24 h before harvest. Quantification of recruited CD11b^highGFP^high cells within aortas was assessed by flow cytometry.

Lungs from mice subjected to CS or air were digested with collagenase as described (Stolberg et al, 2014 (link)) and single cell suspensions were labeled with antibodies for CD11c-V450 (clone N418; eBioscience), CD11b-AlexaFluor 700 (clone M1/70; eBioscience), MerTK-FITC (clone 2B10C42; BioLegend), CD64-APC (clone X54-5/7.1; BioLegend), Ly6C-PE/Cy7 (clone HK1.4; BioLegend), Ly6G-PE (clone 1A8; BioLegend), and CD103-PerCP/Cy5.5 (clone 2E7; BioLegend) at 4°C for 1 h. The population frequencies of resident AMs (CD11b⁻CD11c⁺ or MerTK⁺CD64⁺), neutrophils (CD11b⁺ Ly6G⁺), recruited monocytes (CD11c⁻CD11b⁺Ly6C⁺), and dendritic cells (CD11c⁺CD11b⁻CD103⁺) were analyzed on a BD Fortessa flow cytometer using BD FACSDiva software.

Cell viability was assessed as described above. To assess caspase-1 activation and CD expression, cells were initially washed with 1 ml of PBS, followed by 1 ml of 10% (v/v) FBS in PBS. Cells were then centrifuged and simultaneously stained with 1 x 660-YVAD-FMK (FLICA 660 Caspase-1 assay kit, ImmunoChemistry Technologies) for inflammatory caspase activation and with fluorochrome-conjugated antibodies CD45-BV421 (clone 30-F11), CD11b-PE/Cy5 (clone M1-70) and Ly-6G-PE (clone 1A8) (BioLegend) and CD16-FITC (clone AT154-2, Bio-Rad) in RPMI 1640 medium for 30 min at RT. Cells were washed with 10% (v/v) FBS in PBS and analysed via flow cytometry.