Fluorescein isothiocyanate conjugated cd14 cd14 fitc

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.

Granulocytic and monocytic differentiation analysis was carried out as previously described [6 (link),39 (link)]. Briefly, U937 cells, treated with and without 2.5 mg/mL of PAE, were harvested and 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 were resuspended with 10 µL PE- or FITC-conjugated mouse IgG1 (Pharmingen). Then, samples were incubated for 30 min at 4 °C in the dark, washed in phosphate buffered saline (PBS), and resuspended in 500 µL PBS containing 2 µL propidium iodide (PI) (Sigma-Aldrich). Samples were analyzed by FACS Calibur flow cytometer with Cell Quest technology (Becton Dickinson, San Diego, CA, USA). PI-positive cells were excluded from the analysis.
For each measurement, triplicate analyses were conducted for each sample. Differences between the means were evaluated with analysis of variance (ANOVA), using the InStat 3.0 statistic program (GraphPad Software, San Diego, CA, USA).