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. 2021 Feb 17;218(4):e20200472. doi: 10.1084/jem.20200472

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© 2021 Suwankitwat et al.

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Figure 4. — Myeloid-specific Hem1 disruption impairs neutrophil and monocyte migration. (A–E) Live-cell 2D imaging of BM-derived neutrophils isolated from LysMCre^+/+ and Hem1^fl/fl LysMCre^+/+ mice in response to LTB4 or PBS stimulation for 1 h. 40 cells were tracked per chamber. Images and bar graphs show individual cell migration (A), migration velocity (B), total distance traveled (accumulated = total distance; Euclidean = straight-line distance between start and stop points; C), FMI (D), and directness of migration (E). (F and G) In vivo neutrophil migration to BALF in response to o.p. 10-µg LPS administration. (F) Neutrophil number in BALF before and 2 h after o.p. LPS (left) and Diff-Quik staining of BALF cytospin (right). Scale bars, 100 µm. (G) Neutrophil number in PB before and after o.p. LPS. (H) Diagram depicting an in vitro competitive adult-derived monocyte Transwell migration assay between tdTomato⁻ WT and tdTomato⁺ Hem1^fl/flLysMCre⁺ monocytes in response to 4 h of CCL2 stimulation. Bar graph shows the percentage of migrated cells (stimulated versus unstimulated = migration index). (I) In vivo monocyte migration to BALF in response to o.p. LPS administration. Monocyte number (singlet gated, CD45.2⁺CD11b⁺Ly6G⁻Ly6C⁺) in BALF (left) or PB (right) 24 h after o.p. PBS or LPS. (J) Diagram of chimera BM monocytes of mice with Spn infection (left) and percentage of CD11b⁺Ly6C^hi monocytes in blood, spleen, and BALF of WT and Hem1^fl/flLysMcre⁺ cells. Data shown are mean ± SEM from n = 3–5 mice (6–12 wk old)/genotype, ≥2 independent exps. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; Student’s two-tailed t tests. PMNs, polymorphonuclear cells.