Differentiation of ESC-derived progenitors to neutrophils. (A) Wright-Giemsa staining of mEB8-ER (top) or mBB8-ER cells (bottom) 6 days after the addition of 2 ng/mL G-CSF. (B) Surface expression of Gr-1, CD11b, CD16, and CD80 in mEB8-ER (top) and mBB8-ER cells (bottom) assessed with flow cytometry. Cells were induced to differentiate for 6 days. Cells before induction of differentiation (“0 day”) were used as a negative control (“Control”). (C) Analysis of intracellular [Ca²⁺] in mEB8-ER and mBB8-ER–derived neutrophils in response to fMLP stimulation. Fluorescence was recorded using SpectraMax M2 spectrometer (excitation, 340-380 nm; emission, 510 nm). Results are plotted as emission ratio versus time. Arrow indicates the time of addition of fMLP (100nM). A representative experiment from 5 separate experiments. (D) Migration in the transwell assay of mEB8-ER and mBB8-ER–derived neutrophils. Left: Representative images of cells that crossed the transwell membrane with or without the gradient of fMLP (100nM). Right: The count of cells that crossed the transwell membrane. Each bar represents the mean ± SEM (error bars). *P < .05. All values were normalized to the level (= 1) in cells without fMLP stimulation.