Chemoattractants induce strong cell polarization associated with PLD1 and PLD2 staining. (A) Single labeling. Neutrophils, at a density of 5 × 10⁶ cells/mL, were adhered for 20 minutes to microscope slides in 6-well tissue culture plates. They were then incubated for 14 minutes at 37°C in 5% CO₂ with chemokines, added to the upper part of the slide (more detail in Figure 7). After this, cells were fixed and immunostained with anti-PLD1 or anti-PLD2 antibodies. They were further treated with FITC-conjugated secondary antibodies, and finally observed by confocal fluorescence microscopy. (B-C) Triple labeling. Cells were treated as above but processed with anti-PLD1 (B) or anti-PLD2 antibodies (C), FITC-secondary Ab, TRITC-phalloidin, and DAPI, in sequential order, and were observed by epifluorescence microscopy.