(A) MKs and marrow cells were co-cultured in the presence of 1μM nocodazole, latrunculin A, cytochalasin D, or control vehicle. (B) MKs or marrow cells were treated with latrunculin A or cytochalasin D for two hours. After thorough washings, cells were co-cultured with untreated marrow cells or MKs, respectively. (A-B) Cells are stained with anti-CD41, -CD18 or -Ly6G and Draq5, and observed by confocal microscopy. Histograms show percentages of MKs containing at least one neutrophil. At least 150 (A) or 500 (B) MKs per condition were counted; pool of 3 independent experiments (See Figure 3—source data 1) (C) Cells are stained with anti-CD41 (green), anti-CD18 (blue) and phalloidin (red). DNA is visualized with Hoechst (gray). Images show F-actin on MK surface where neutrophils are attached (upper photos), around neutrophils encapsulated in CD41+ vacuoles (middle photos) or free within MKs (lower photos). (D-G) Emperipolesis assay was performed (A) in the presence of 10μg/ml anti-CD18 or corresponding isotype control rat IgG1 (B) using marrow cells from WT versus CD18-deficient mice or (C) using MKs from WT versus ICAM-1-deficient mice, or (D) in the presence of 1μM of ezrin inhibitor NSC668394. (A-D) Histograms show percentages of MKs containing at least one neutrophil. At least 350 MKs per condition were counted; pool of 2 (F), 3 (E), or 4 (D and G) independent experiments. (See Figure 3—source data 1) H. After co-culture, cells are stained with anti-CD41 (white), anti-ezrin (green) and anti-Ly6G (blue). Arrows show ezrin clustering on the MK surface. Red asterisks show MKs without detectable ezrin. (I) Cells are stained with anti-CD41 (gray), -ezrin (green), -ICAM-1 (red), -Ly6G (blue). Arrows show ICAM-1/ezrin co-localization on the MK surface. Lower photo is a magnification of the dashed white region. (Hand I). Scale bars represent 20μm, representative of at least 3 independent experiments.