title: Live cell imaging of paxillin in rolling neutrophils by dual-color quantitative dynamic footprinting.
abstract: 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.
authors.affiliation: Division of Inflammation Biology, La Jolla Institute for Allergy and Immunology, La Jolla, California, USA.
authors.forename: Prithu
authors.initials: P
authors.lastname: Sundd
authors.forename: Edgar
authors.initials: E
authors.lastname: Gutierrez
authors.forename: Brian G
authors.initials: BG
authors.lastname: Petrich
authors.forename: Mark H
authors.initials: MH
authors.lastname: Ginsberg
authors.forename: Alex
authors.initials: A
authors.lastname: Groisman
authors.forename: Klaus
authors.initials: K
authors.lastname: Ley
mesh_terms: D000818:Animals; D002232:Carbocyanines / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D036904:Leukocyte Rolling / Q000502:physiology*; D051379:Mice; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D009504:Neutrophils / Q000166:cytology / Q000378:metabolism*; D051419:Paxillin / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D002232:Carbocyanines; D005456:Fluorescent Dyes; D051419:Paxillin; C496062:Pxn protein, mouse; D011993:Recombinant Fusion Proteins; C024286:3,3'-dioctadecylindocarbocyanine