Fig. 3
Neutrophil-like dHL-60 and adhesion to endothelial monolayer. A Left bar plot shows mean ± SD of GLP1 receptor and CD11b myeloid maker mRNA expression levels on neutrophil-like dHL-60 cells. Their mRNA expression levels were quantified by real time PCR and represented according to β-actin expression levels as arbitrary units (a.u.) based on 2-(ACTB/gene) algorithm. Different regions of the GLP1R (NM_002062.5) were checked. Right bar plot with individual points represents the CD11b levels on dHL-60, with/without fMLP treatment at 10 μM for 90 min, by flow cytometry, expressed in relative fluorescence units (RFU). The CD11b levels were upregulated with statistical significance after fMLP treatment [n = 6, CONTROL (195 ± 45.53) vs fMLP (207 ± 47.24); t(5) = 3.985, p = 0.0105; p < 0.05]. We also checked this upregulation by performing an immunocytofluorescence using an anti-human CD11b-PE antibody (Scale bar = 25 μm). B Neutrophil-like dHL-60 cells migration towards fMLP stimulus on a Sun-Chip. The neutrophils tracer and migration were detected by a ChemiDoc MP Imaging System after their fluorescence staining with calcein-AM. This method was validated for analysing the correlation between relative fluorescence units (R.F.U.) and number of cells [R2 = 0.9862, F(1,2) = 142.7, p = 0.0069; p < 0.05]. C fMLP-treated neutrophil-like dHL-60 cells adhesion on human aortic endothelial (HAEC) monolayer previously exposed to normal (NG) or high glucose (HG) for 30,120 and 360 min. The subtraction of basal and after washing the non- adhered and stained neutrophils to HAEC were quantified by ChemiDoc MP Imaging and represented as percentage over basal. A workflow of this process is shown. Graph represents the paired data regarding NG and HG. The statistical analysis showed differences on 30min treatment [n = 5, NG 0.5 h (79.58 ± 6.75) vs HG 0.5 h (95.46 ± 4.13); t(4) = 4.469, p = 0.0111; p < 0.05] and 120 min [n = 5, NG 2h (79.39 ± 10.49) vs HG 2h (87.65 ± 6.916); t(4) = 2.857, p = 0.046; p < 0.05]. Validation was also performed by fluorescence microscopy images (HAEC are shown in red, whereas dHL-60 cells, in green. Scale bar = 100 μm)