Figure S1.
A sterile inflammation model in the mouse hind footpad using 2P-IVM.(A) Setup of the mouse footpad inflammation model. Upper panel: Diagram of the 2P-IVM procedure in the LyzM-GFP mouse footpad. Lower panels: Mice were injected with either saline (left panel) or E. coli BPs (right panel) in their foot pads and imaged ∼40–60 min after treatment by 2P-IVM. Maximum-intensity projection of Z-stacks is presented. Vasculature is shown in blue and neutrophils in green. Pink arrowheads mark extravasating neutrophils (right panel). Scale bars = 40 µm. The experiments were performed in n = 2 mice. See Video 1. (B–D) Neutrophils purified from WT mice were stained with CellTracker Green CMFDA, treated with either 40 µM Y27632 or 10 µM nBleb or the vehicle (DMSO) for ∼30 min, adoptively transferred into Alox5−/− mice with inflamed footpads, and imaged by 2P-IVM. Maximum-intensity projections of Z-stacks are presented (B). Magenta arrowheads highlight neutrophils (green) extravasating from the blood vessels (blue). Scale bars = 20 µm. Quantification of the percentage of neutrophils that exhibited arrest (C) or extravasation (D) during the late phase of response, typically 45–90 min after neutrophil transfer to the recipient mice. Data are plotted as mean ± SEM from n = 3 mice for each condition, and each dot represents the result from an individual animal. No significant difference was observed upon analysis with unpaired t test with Welch’s correction. Unpaired t test with Welch’s correction was used to determine statistical significance. (E) Neutrophils purified from GFP-NMIIA mice were treated with either the vehicle (DMSO; top row) or 5 µM MK886 (bottom row), injected into Alox5−/− mice with inflamed footpads, and imaged in time-lapse mode by ISMic. The optical sections were used to quantify the redistribution index of GFP-NMIIA. Scale bars = 3 µm. For each time point, each section in the z axis was given a score of 0 or 1 if the GFP-NMIIA was enriched at the center or cortex, respectively, of an arrested neutrophil manually. An index was generated by averaging the score to the number of stacks analyzed for each neutrophil that displayed arrest behavior during ISMic analysis. For further details, see the Materials and methods section.

A sterile inflammation model in the mouse hind footpad using 2P-IVM. (A) Setup of the mouse footpad inflammation model. Upper panel: Diagram of the 2P-IVM procedure in the LyzM-GFP mouse footpad. Lower panels: Mice were injected with either saline (left panel) or E. coli BPs (right panel) in their foot pads and imaged ∼40–60 min after treatment by 2P-IVM. Maximum-intensity projection of Z-stacks is presented. Vasculature is shown in blue and neutrophils in green. Pink arrowheads mark extravasating neutrophils (right panel). Scale bars = 40 µm. The experiments were performed in n = 2 mice. See Video 1. (B–D) Neutrophils purified from WT mice were stained with CellTracker Green CMFDA, treated with either 40 µM Y27632 or 10 µM nBleb or the vehicle (DMSO) for ∼30 min, adoptively transferred into Alox5−/− mice with inflamed footpads, and imaged by 2P-IVM. Maximum-intensity projections of Z-stacks are presented (B). Magenta arrowheads highlight neutrophils (green) extravasating from the blood vessels (blue). Scale bars = 20 µm. Quantification of the percentage of neutrophils that exhibited arrest (C) or extravasation (D) during the late phase of response, typically 45–90 min after neutrophil transfer to the recipient mice. Data are plotted as mean ± SEM from n = 3 mice for each condition, and each dot represents the result from an individual animal. No significant difference was observed upon analysis with unpaired t test with Welch’s correction. Unpaired t test with Welch’s correction was used to determine statistical significance. (E) Neutrophils purified from GFP-NMIIA mice were treated with either the vehicle (DMSO; top row) or 5 µM MK886 (bottom row), injected into Alox5−/− mice with inflamed footpads, and imaged in time-lapse mode by ISMic. The optical sections were used to quantify the redistribution index of GFP-NMIIA. Scale bars = 3 µm. For each time point, each section in the z axis was given a score of 0 or 1 if the GFP-NMIIA was enriched at the center or cortex, respectively, of an arrested neutrophil manually. An index was generated by averaging the score to the number of stacks analyzed for each neutrophil that displayed arrest behavior during ISMic analysis. For further details, see the Materials and methods section.

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