Granulocytic and Monocytic Differentiation Analysis

Analysis of granulocytic and monocytic differentiation was conducted as previously reported [22 (link),23 (link),24 (link)]. In brief, U937 cells treated with and without 2.5 mg/mL of BGE were resuspended in 10 µL phycoerythrin-conjugated CD11c (CD11c-PE) (Pharmingen, San Diego, CA, USA) or 10 µL fluorescein isothiocyanate-conjugated CD14 (CD14-FITC) (Pharmingen). Controls without BGE treatment were with 10 µL PE- or FITC-conjugated mouse IgG1 (Pharmingen). After incubation at 4 °C for 30 min in the dark, samples were washed in PBS, and resuspended in 500 µL of PBS containing 2 µL of propidium iodide (PI) (Sigma-Aldrich). Analysis of samples was performed by FACS Calibur flow cytometer with Cell Quest technology (Becton Dickinson, San Diego, CA, USA). PI-positive cells were not considered in the analysis. Each data point was the average of individual experiments performed in duplicate, and standard deviation was computed.