JCS020412 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Migration and polarization of neutrophils under basal conditions in the ibidi chemotaxis system. (a) Distance moved by cells in the absence of an fMLP gradient (no fMLP) and in the presence of an fMLP gradient. (b) Eccentricity of unstimulated and fMLP-stimulated neutrophils. *P<0.05 compared to wild-type, Students t-test, n=3 (a), n=minimum of 153 cells (b).

• Supplemental Figure S2 -

  Fig. S2. Time-lapse sequence of a single neutrophil demonstrating the analysis of polarization, orientation, speed and chemotactic index. The time since fMLP addition is recorded in the upper-right corner of each panel (in minutes) and the source of fMLP is located off-screen at the top of the image. (a) Polarization is determined by measuring the eccentricity of the cell. The major axis (long axis, red) and the minor axis (green) are measured. Polarized cells have an eccentricity (major axis/minor axis) of &γτ;0.2; unpolarized cells have an eccentricity of <0.2. (b) A cell is considered orientated towards the chemoattractant if the angle between the long axis and chemoattractant is &λτ;45°. (c) Cells undergoing chemotaxis tend to be both polarized and orientated towards the chemoattractant. (d) The centroid position (blue circles) of the cell at each time point is recorded for every frame. (e) The total distance the cell migrated during the experiment (Σ_dist, blue line) can be calculated by measuring the distance between each recorded centroid, whereas the displacement (Δ_x, black line) can be calculated by measuring the distance the cell moved towards the chemoattractant. Speed is equal to Σ_dist/duration of experiment; chemotactic index is equal to Σ_dist/Δ_x.