Phycoerythrin pe conjugated anti mouse cd86

LiChroslov hypergrade acetonitrile and formic acid were purchased from Sigma-Aldrich (Schnelldorf, Germany), and water for UHPLC analysis was purified using a Milli-Q Plus system (Merck Millipore, Burlington, MA, USA). Reference compounds chlorogenic acid, rosmarinic acid, and caffeic acid were purchased from Sigma-Aldrich (Schnelldorf, Germany). All reference flavonoid compounds were acquired from PhytoLab (Vestenbergsgreuth, Germany). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2,2-Diphenyl-1-picrylhydrazyl (DPPH), Folin-Ciocalteu reagent, sodium carbonate (Na₂CO₃), gallic acid, L-ascorbic acid, aluminium trichloride (AlCl₃), quercetin, foetal bovine serum (FBS), Hank’s buffered saline solution (HBSS), and trypsin, lipopolysaccharide (LPS) were obtained from Sigma Aldrich. RPMI-1640 medium with Glutamax was produced by Gibco. Mouse monocyte-colony stimulating factor (M-CSF) and interferon-gamma (IFN-γ) were obtained from PeproTech (London, UK). FITC-conjugated anti-mouse F4/80, phycoerythrin (PE)-conjugated anti-mouse CD86, and biotin-conjugated anti-mouse CD80 antibodies were purchased from BioLegend (San Diego, CA, USA).

BMDMs were incubated with PSRE and PACN (100 μg/mL each) and LPS + IFNγ (10 ng/mL/100 U/mL) for 24 h. The cells were washed twice with HBSS and harvested by trypsin (0.5%), then DMEM-high glucose medium with 10% FBS was added and cell suspension was centrifuged at 300× g for 5 min. Then cells were incubated with specific conjugated antibody mixtures (in concentration 1:100 in cell wash buffer) for 30 min on ice in the dark. The mixture contained following monoclonal antibodies purchased from BioLegend (San Diego, CA, USA): FITC-conjugated anti-mouse F4/80, phycoerythrin (PE)-conjugated anti-mouse CD86 and biotin-conjugated anti-mouse CD80. Then cells were washed and stained with streptavidin-APC-Cy7. After 30 min, cells were washed and stained for evaluation for apoptosis (see method above). After staining samples were analyzed by flow cytometry (BD FACS Melody™, BD Biosciences, San Jose, CA, USA).