Fig. 2

Replacing serum with Nutridoma during differentiation increases FPR1 surface expression and chemotactic efficiency. PLB-985 cells were differentiated into a neutrophil-like state by culturing in media supplemented with 1.3% DMSO and 9% FBS or supplemented with 1.3% DMSO, 2% Nutridoma and 0.5% FBS, for 6 days. Then cells were stained with FLPEP (a) or an antibody against CD11b (b) and measured by cytometry. Data was analyzed using MATLAB. The experiment was repeated three times and a representative experiment is shown. (c) Cells differentiated as in (a) and (b) were plated under agarose and analyzed in an automated chemotaxis assay by time-lapse microscopy with chemoattractant uncaging of Nv-fMLF. A cell directionality parameter measuring the angular bias of cell movement towards the gradient source is shown. This experiment was performed three times. Error bars represent the standard error of the mean. Images and statistics were processed using custom MATLAB software. Asterisk indicates a p-value of less than 0.05 using a t-test. d Cells differentiated as in (a) and (b) were mixed in suspension with pHrodo Green-labeled dead Staphylococcus aureus bioparticles for 2 h at 37 degrees. Phagocytosis of the particles was then analyzed by cytometry. e PLB-985 cells were differentiated into a neutrophil-like state as in (a) and (b). Cells were then incubated with NBT solution and 100 ng/mL of PMA or 1 μM fMLF, at 37 °C for 15 min, and measured by cytometry