1. Visualizing the Neutrophil Response to Sterile Tissue Injury in Mouse Dermis Reveals a Three-Phase Cascade of Events 
Lai Guan Ng, Jim S. Qin, Ben Roediger, Yilin Wang, Rohit Jain, Lois L. Cavanagh, Adrian L. Smith, Cheryl A. Jones, Michael de Veer, Michele A. Grimbaldeston, Els N. Meeusen, Wolfgang Weninger 
Journal of Investigative Dermatology 
Volume 131, Issue 10, Pages (October 2011)
DOI: /jid
Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Experimental models for the study of neutrophil migration in the dermis of mice. (a) Figures depict intravenous and intradermal neutrophil transfer models. (b) Representative time-lapse images from multi-photon imaging showing the migratory behavior of neutrophils in the dermis of mice following intravenous or intradermal transfer. Red box represents laser injury site. (c) Time-lapse images showing the migratory pattern of intradermally transferred neutrophils in the ear skin at resting state and after tissue injury. (d) Vmean and meandering index (MI) of neutrophils transferred intravenously (open circle, after injury) or intradermally (open square, resting; open triangle, after injury). White lines represent migratory tracks. Bar=59μm. Time scale, min:sec. HF, hair follicle; ID, intradermal; IV, intravenous.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Neutrophil responses to tissue injury occur in three sequential phases. (a) Representative time-lapse images showing neutrophil responses to injury. Arrows illustrate attraction of scouting neutrophils to the injury site. Dotted line represents a reference line for orientation purposes. Bar=59μm. (b) High-magnification images depicting neutrophils at the injury site during scouting and amplification phases. (c) Graph shows neutrophil distance to the injury site over time. Each line represents an individual neutrophil track. (d) Volumetric measurement of neutrophil clusters at the injury site over time. Data were obtained from three independent experiments. Bars represent mean±SEM. (e) Comparison of the meandering index (MI), displacement rate, and Vmean of neutrophils during different phases. Time scale, min:sec. As indicated in the table, the analysis of variance test was performed for comparison between groups.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Myelomonocytic cell migration in the dermis of mice. (a) Figures depict a skin injury model induced by needle scratching. (b) Representative time-lapse images from multi-photon imaging showing the migratory behavior of green fluorescent protein (GFP)+ cells in the dermis of Lys-GFP mice (on the C57BL/6 background) following needle scratching. Dashed lines represent needle injury site. (c) Frequency of GFP+ cells attracted to the site over the period of 2h after needle scratch injury. Numbers indicate each individual step of the cell response. This dataset was obtained from a representative mouse out of three. (d) Vmean and MI of GFP+ cells attracted to the injury site during steps 2 and 3. The dataset shown is representative of three independent experiments. Bar=100μm. Time scale, min:sec. HF, hair follicle.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Migratory mechanisms of neutrophils in response to tissue injury. (a) Left panel, representative time-lapse images showing neutrophil responses to injury after pertussis toxin (PTX) pretreatment. Right panel, comparison of the Vmean and MI of PTX-pretreated neutrophils before and after injury. (b) Representative time-lapse images showing neutrophil reponses to injury after 8-bromo-cyclic ADP ribose (8-BR) pretreatment. Red box represents injury site. Small insets show high magnification of neutrophils at the injury site. Bar=59μm. Time scale, min:sec. (c) Left panel, frequency of scouting neutrophils attracted to the injury site after 8-BR pretreatment. Right panel, comparison of the Vmean and MI of control and 8-BR-treated neutrophils after tissue injury. (d) Volumetric measurement of neutrophil clusters at the injury site after transfer of control and 8-BR-treated neutrophils. Data were obtained from at least three independent experiments. Bars represents mean±SEM.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
Neutrophils are present in non-inflamed skin in mice and sheep. (a) Flow cytometric analyses of neutrophils in normal mouse ear skin after IV injection of 0.7μg Gr-1-PE antibody (see Supplementary Figure S7 online). Dot plots depict Ly6G and Gr-1 staining of single-cell suspensions prepared from the blood or skin. Graph shows the frequency of Gr-1-PElowLy6G+ cells. (b) Whole mount ear skin preparation from mice injected IV with isolectin-Alexa647, and stained with Ly6G followed by anti-rat Alexa488. Bar=39μm. (c) Left panel, a snapshot of lys-ablGFP mouse ear skin from mice injected intravenously with Evans blue. Right panel, display of the same image in blend mode to illustrate extravascular GFP+ cells in the skin. Bar=100μm. (d) Vmean and meandering index (MI) of extravascular GFPbright cells under homeostatic conditions. (e) Frequency of neutrophils detected in the lymph collected from the pseudo-afferent lymphatics in a sheep model. Left panel shows numbers in resting lymph (open circles), whereas the right panel shows numbers of neutrophils at 6hours following injection of ovalbumin (Ova) (250μg) in alum (open triangles). Symbols represent individual pseudo-afferent sides. (f) Graph shows the number of carboxyfluorescein succinimidyl ester–labeled neutrophils (per 10,000 cells) and lymphocytes (per 500,000 cells) detected in resting lymph at various times after the intravenous injection.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions