Cd11b clone icrf44

After 18 h, supernatants were collected and cells detached using ice-cold PBS-EDTA, and stained using the following conjugated antibodies for surface markers: CD14 (clone M5E2, Biolegend), CD16 (clone NKP15, BD Biosciences), CD25 (clone M-A251, BD Biosciences), CD11b (clone ICRF44, Biolegend), CD80 (clone L307.4, BD Biosciences), CD69 (clone FN50, Biolegend). Dead cells were excluded with with Fixable Viability Dye BV421 (BD Biosciences). Samples were run on a BD Fortessa flow cytometer and data analysed using FlowJo software (BD Biosciences).

Macrophage precursors were plated at 1 × 10⁶ cells/well in 6-well plates and differentiated in macrophage medium for a week. Macrophages were lifted from 6-well plates by incubation with StemPro Accutase (Gibco) for 10 min at 37 °C. The cells were washed with PBS and non-specific binding sites were blocked by incubation in FACS buffer (PBS, 1% FCS, 10 μg/mL human IgG) for 10 min at RT. 2 × 10⁵ cells per sample were stained with directly-conjugated primary antibodies against CD11b (clone ICRF44, Biolegend), CD14 (clone 18D11, Immunotools) and CD45 (clone MEM-28, Immunotools), for 30 min at RT. Cells were then washed twice with FACS buffer and fixed with 4% paraformaldehyde (PFA) for 10 min at RT. Cells were washed with PBS and analysed on a FACS Calibur flow cytometer (BD Biosciences). Fluorophore-conjugated isotype controls from the same manufacturers were used.