Fitc a anti cd14

We recruited 9 healthy donors ranging from 26 to 40 years, 5 man and 4 women, from our facility. A medical anamnesis was performed by an internal medicine specialist before whole blood extraction. No medication uses nor relevant past medical history were reported. The ethics committee of Facultad de Medicina de la Universidad de Chile approved the study protocol. 2.2.Whole blood lysis protocol and flow cytometry. Venous blood (30ml) was obtained by cubital venopunction from all participants. PBMC Ficoll extraction was performed as previously dercribed(REF). PBMC were stained in duplicate with the following antibodies: FITC-A-anti-CD14, APC-H7-anti CD16, PERCP-Cy5.5-anti-CD86, APC-A-anti-CD16, V450-A-anti-IL-1β (Biolegend, US) at room temperature for 30 minutes. 1ml of BD FACS Lysing Solution (BD Biosciences) at 1x concentration was used for erythrocyte lysis. Finally cells we fixed and permeabilized using a fixation/permeabilization BD Kit (BD Biosciences).

To phenotype monocyte subsets, 1 × 10 6 /cells per tube were stimulated in duplicate with lipopolysaccharide, (1 µg/mL) (BD Biosciences, NY) and IL-4 (200 U/ml) (BD Biosciences, NY) in RPMI 1640 (BD Biosciences, NY) medium mixed with 10% fetal bovine serum (BD Biosciences, NY) for 2 h and 4 h respectively at 37°C in 5% CO2 and exposed to brefeldin A (BD Biosciences) as described in previous studies. Then, the cells were washed, followed by staining with V710-anti-CD3 (Biolegend, US), V650-anti-CD19, (Biolegend, US), FITC-A-anti-CD14, APC-H7-anti CD16, PERCP-Cy5.5-anti-CD86, APC-A-anti-CD16, (Biolegend, US). Subsequently, they were fixed and permeabilized and stained with V450-A-anti-IL-1β (Biolegend, US). The frequencies monocyte subsets were assessed on a FACS Verse instrument (BD, Franklin Lakes, US), and the data was analyzed with FlowJo software (v7.6.1; TreeStar; Ashland, US). 2.4.Statistical Analysis. Variables were shown as median and range values. We employed the Mann-Whitney U nonparametric test to evaluate the differences among groups. The relationship between variables was analyzed by the Spearman rank correlation test. All of the data were carried out with the GraphPad Prism version 6.01 software. P value of <0.05 represented statistical significance.