Directionality analysis of PLD1 and PLD2. (A) Experimental setup. IL-8 was added at a precise location (red dot) with a 200-μm micropipette to a microscope slide of adherent neutrophils to form a temporary gradient prior to anti-PLD2 immunostaining. (B) Cells exhibiting polarization caps that were pointing within a 90° swath toward IL-8 were counted, and the percentage over total number of cells was calculated. Results are the mean ± SEM from 3 independent experiments conducted in duplicate. An example of one such field, observed by epifluorescence microscopy, is presented in panel C. The yellow lines (added with Adobe Illustrator software, v. 11.0; San Diego, CA) were defined as intersecting the main polarization cap (bright PLD2 immunostaining) of the cells in the direction of the chemokine.