Objective: Neutrophil recruitment to sites of inflammation involves P-selectin-dependent rolling. qDF is a useful tool to visualize the topography of the neutrophil footprint as it interacts with the substrate. However, elucidating the role of specific proteins in addition to topography requires simultaneous visualization of two fluorochromes.
Methods: To validate DqDF, mouse neutrophils were labeled with the membrane dyes DiO and DiI and perfused into microchannels coated with P-selectin-Fc. Footprints of rolling neutrophils were recorded as two separate images, one for each fluorochrome. To assess the localization of the cytoskeletal protein paxillin, we applied DqDF to DiO-stained neutrophils of mice expressing an mCherry-paxillin fusion protein.
Results: The footprint topographies obtained from DiO and DiI in the plasma membrane were identical. The z-coordinates of the microvilli tips obtained with the two fluorochromes in the footprint were also identical. Paxillin was found to be localized to some, but not all ridges in the neutrophil footprint.
Conclusions: Our data suggest that the spectral properties of the fluorochrome do not affect the results. DqDF will be useful for simultaneous visualization of two fluorochromes in the footprint of rolling cells.
© 2011 John Wiley & Sons Ltd.