Flow Cytometric Analysis of HMDM Phenotypes

Flow cytometric analysis was performed as described previously [10 (link),32 (link)]. Briefly, after 48 h of treatments, HMDMs were washed, detached in 2.5 mM EDTA solution on ice and blocked with BD Fc Block™Pure (564220, BD Biosciences, San Jose, CA, USA) for 10 min at room temperature. HLA-DRPerCP-Cy5.5 (552764; Clone: G46-6, BD Biosciences), CD80 BB51 (565008; Clone: L207.4, BD Biosciences), CD163PE-CF594 (562670; Clone: GHI/61, BD Biosciences) or CD14 APC (555399; Clone: M5E2, BD Biosciences) antibodies were added in each sample and incubated on ice for 30 min according to manufacturers’ suggestions. HMDMs were washed and fixed with ice-cold 1% PFA solution, stored on ice, and analysed with a BD LSRFortessa™ Cell Analyzer and the BD FACSDiva v8.0.1 software (BD Biosciences). To quantify the surface marker expression, median fluorescence intensities of singlet cells were used.