Visualization of EP in MKs of different maturational stages. Hematopoietic progenitor cells were cultured in TPO medium for 1, 2, or 4 days to obtain MKs of different maturation levels. MKs were then cocultured with bone marrow cells for 12 hours. MKs were stained with anti-CD41 AF488 (green), neutrophils were stained with anti-Ly6G AF594 (red), and DNA was stained with Hoechst 33342 (blue). MK maturity was graded based on the extent of the DMS (early, intermediate, and late DMS). Images were obtained using a Zeiss LSM 800 with Airyscan attached to a Zeiss Axio Observer Z1 Inverted Microscope with a Plan-Apochromat 63x objective. Scale bars, 10 µm. (A) Immature MK showing DMS beginning to develop between the nuclear lobes and forming connections with the MK surface (early DMS). (B) With increasing maturation, the DMS becomes more prominent and forms thicker connections with the MK surface (intermediate DMS). (C) Mature MK with extensive DMS occupying the majority of the MK cytoplasm (late DMS). (D-F) Early, intermediate, and late DMS MKs engulfing neutrophils during EP. (G) EP frequency across MK maturational stages. 100 MKs per maturational stage per experiment were counted in each of 3 independent experiments. (H) The number of engulfed neutrophils per EP event across MK maturational stages. Pooled data from 3 independent experiments (n = 300 MKs per maturational stage; EP events: early DMS MKs: 1, intermediate DMS MKs: 13, and late DMS MKs: 61). (I) Z-projection of mature MK (late DMS) containing 11 neutrophils (*s). (J) EP assay with neutrophils isolated from blood or bone marrow; 4 independent experiments. (K) EP assay with peritoneal cells harvested 2 hours after IP injection of 25 ng/mL IL1B. Green, CD41; red, Ly6G; blue, CD18; gray, DNA. Scale bars, 10 µm.