Cd80 bv650

Primary antibodies against LAMP1, LAMP2, Cathepsin D, Cathepsin L, p62, galectin-3 and secondary goat anti-mouse IgG (Alexa Fluor 647) were purchased from Abcam (Cambridge, UK). LC3A/B was from Cell Signaling (Bioke, Leiden, The Netherlands), actin-HRP from Santa Cruz Biotechnology (Santa Cruz, CA, USA), CD86-Alexa700 and HLA-DR-PeCy5 from BD Biosciences (Erembodegem, Belgium) and CD80-BV650, CD14-FITC and CD163-Alexa647 were bought from Biolegend (ITK diagnostics, Uithoorn, The Netherlands). NDP52 (CALCOCO2), secondary goat anti-rabbit IgG (Alexa Fluor 647) and HRP-conjugated antibodies reactive with mouse and rabbit were purchased from Thermo Fisher Scientific (Merelbeke, Belgium).

To examine surface marker expression, BMDMs were detached using citrate buffer (17 mM tri-Sodium citrate dehydrate and 135 mM potassium chloride in water), transferred to V-bottom well plates, incubated with 1:50 anti-CD16/CD32 Fc-block and stained for 20 min at room temperature in the dark with antibodies. Antibodies were directed to CD40-APC (Biolegend, 1:100), MHC-II-PerCP/Cy5.5 (Biolegend, 1:200), CD80-BV650 (Biolegend, 1:100) and CD86-BV510 (Biolegend, 1:100) for LPS-stimulated macrophages or directed to CD71-PE (BD PharMingen, 1:250), CD206-APC (Biolegend, 1:250), CD273-PE (BD PharMingen, 1:250), CD301-AF647 (Serotec, 1:250) or isotype controls (Biolegend) for IL-4 stimulated macrophages (IGG2A-PE, IGG2A-APC), diluted in staining buffer (PBS with 0.5% BSA and 2.5 mM ethylenediaminetetraacetic acid; EDTA). After staining, cells were washed and resuspended in staining buffer, measured on a Beckman Coulter CytoFLEX or BD LSR Fortessa and analyzed using FlowJo (Tree Star). Surface expression was calculated as ΔMFI = (median fluorescence intensity)_{positive staining} – (median fluorescence intensity)_{isotype or FMO control}. MFI controls (isotype or FMO) were measured on a pool of all samples.