Distribution and dynamics of neutrophils in tissues. (A) Experimental scheme and imaging of Ly6GTOM neutrophils in tissues of the WT parabiotic partner. (B) Distribution of neutrophils in optically cleared organs by multiphoton microscopy (for BM; white is bone captured by second harmonic generation) or LSM; n = organs from 2–3 mice. Blue, vessels; green, host-derived neutrophils; red, partner-derived neutrophils. Bars, 500 µm. See also Videos 1–8 showing partner-derived neutrophils within the tissue. (C) Positioning plots showing the distribution of host- (blue) and partner-derived (red) neutrophils in two-dimensional maps of each tissue. Arrows show areas where neutrophils cluster. Plots obtained from the tissues shown in B. (D) Confocal reconstruction of neutrophils (red) proximal to blood vessels (green) in representative tissues from parabionts as in A. Partner-derived neutrophils were preferentially found in the parenchyma (extravascular) or within vessels (intravascular), depending on the tissues, as quantified in the right bar graph. Bars show mean ± SEM values from the analysis 31–73 neutrophils per tissue from three different animals. (E) Left, experimental design. Curves show the number of partner-derived neutrophils as determined by flow cytometry from the tissues of parabiotic pairs collected at the indicated ZTs. Values are normalized to ZT5 and duplicated over two complete days for better visualization. Periods of darkness are shown by the shaded rectangles, and the red dotted lines show the oscillations in blood for reference. Values are mean ± SEM from 10–12 mice per time and tissue from two experiments. Intestine refers to the large intestine (colon).
