Figure 4.

G-CSF inhibits the steady increase in cytosolic free Ca²⁺ during neutrophil aging, which is essential for apoptosis. Neutrophils were loaded with the fluorescent Ca²⁺ indicator Fluo-3 and incubated in the presence or absence of 10 ng/mL G-CSF in the stirred cuvette of a fluorometer, while the increase in fluorescence was measured every minute for 12 hours. As controls, cells where loaded with 10 μM of the cell permeable Ca²⁺-chelator BAPTA-AM or incubated in the absence of extracellular Ca²⁺ (A). Data represent the average of 3 independent experiments. An increase in intracellular free Ca²⁺ is a requirement for the process of neutrophil apoptosis, because treating the cells with an increasing dose of BAPTA-AM induced up to 75% neutrophil survival at 10 μM after 18 hours of incubation (B). The percentage of surviving cells is expressed as a lack of annexin V/PI staining determined by flow cytometry. Data represent the mean (± SEM) of 3 independent experiments performed in duplicate.