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. Author manuscript; available in PMC: 2016 Nov 1.

Published in final edited form as: J Immunol Methods. 2015 Sep 2;426:120–127. doi: 10.1016/j.jim.2015.08.012

Fig. 3 — (A) Neutrophils from CD11b-mYFP homozygous mouse before and after activation on ICAM-1. Fluorescent and bright field images on the left show CD11b-mYFP neutrophils without stimulus at room temperature and those on the right show a CD11b-mYFP neutrophil at 37 °C in the presence of fMLP. (B) Neutrophils from CD11b-mYFP homozygous mouse were allowed to migrate on an ICAM-1-coated coverslip in the presence of fMLP and fluorescence of migrating neutrophils was recorded. A fluorescent image from each video was presented (left), and the YFP intensity over the cell body of the indicated neutrophils was alternatively shown in rainbow colors, highest (red) to lowest (purple), along the dotted line from the rear end to the front (center) or in graphs (right). The area marked with a white line in the center figures indicates lamellipodia. Scale bar, 10 μm. (C, D) CD11b-mYFP⁺ cell migration in vivo. Intravital two-photon microscopy was performed to observe migration of CD11b-mYFP⁺ cells in cremaster vasculature inflamed with fMLP (C) and in the trachea infected with influenza virus (D). Green, YFP-positive cells; Red, blood. Scale bar, 20 μm. Each curved line in (D) indicates the migratory track of the individual YFP-positive cell for 15 min. (E) CD11b-mYFP⁺ cell infiltration into infected tissue. CD11b-mYFP homozygous mouse was infected with influenza virus, and infiltrating leukocytes in the lung at day 4 post-infection were examined using flow cytometry. CD11b-mYFP-positive leukocytes (YFP⁺CD45.2⁺) were divided into two groups based on YFP fluorescence intensity, YFP^intermediate (red) or YFP^high (blue), and the identity of each group was shown by staining with antibodies to the indicated cell type-specific surface markers.

Fig. 3 — (A) Neutrophils from CD11b-mYFP homozygous mouse before and after activation on ICAM-1. Fluorescent and bright field images on the left show CD11b-mYFP neutrophils without stimulus at room temperature and those on the right show a CD11b-mYFP neutrophil at 37 °C in the presence of fMLP. (B) Neutrophils from CD11b-mYFP homozygous mouse were allowed to migrate on an ICAM-1-coated coverslip in the presence of fMLP and fluorescence of migrating neutrophils was recorded. A fluorescent image from each video was presented (left), and the YFP intensity over the cell body of the indicated neutrophils was alternatively shown in rainbow colors, highest (red) to lowest (purple), along the dotted line from the rear end to the front (center) or in graphs (right). The area marked with a white line in the center figures indicates lamellipodia. Scale bar, 10 μm. (C, D) CD11b-mYFP⁺ cell migration in vivo. Intravital two-photon microscopy was performed to observe migration of CD11b-mYFP⁺ cells in cremaster vasculature inflamed with fMLP (C) and in the trachea infected with influenza virus (D). Green, YFP-positive cells; Red, blood. Scale bar, 20 μm. Each curved line in (D) indicates the migratory track of the individual YFP-positive cell for 15 min. (E) CD11b-mYFP⁺ cell infiltration into infected tissue. CD11b-mYFP homozygous mouse was infected with influenza virus, and infiltrating leukocytes in the lung at day 4 post-infection were examined using flow cytometry. CD11b-mYFP-positive leukocytes (YFP⁺CD45.2⁺) were divided into two groups based on YFP fluorescence intensity, YFP^intermediate (red) or YFP^high (blue), and the identity of each group was shown by staining with antibodies to the indicated cell type-specific surface markers.