G-CSF inhibits neutrophil apoptosis. Neutrophils were incubated in the absence or presence of G-CSF (10 ng/mL). Samples were taken at various times to determine the indicated hallmarks of apoptosis. (A) Cells were stained with FITC-labeled annexin V and PI and analyzed by flow cytometry. The percentage of double negative cells is expressed as survival. (B) Cells were loaded with the ▵ψ_m-sensitive dye JC-1 and analyzed by flow cytometry. When JC-1 accumulates in mitochondria with a high ▵ψ_m it displays red fluorescence (FL-2), while the dye is green fluorescent (FL-1) when ▵ψ_m is low. The percentage of cells with a high ▵ψ_m is expressed in this graph, corrected for background staining (ie, the JC-1 fluorescence of cells treated with 1 μM of the uncoupler CCCP). The average slope for the loss of ▵ψ_m in the control cells was −3.53 plus or minus 0.1801, SEM) %/h, while the average slope for the G-CSF–treated cells was −2.66 plus or minus 0.1927, SEM) %/h. The difference between the slopes is highly significant with a P value of .001. (C) Cells (2 × 10⁵) were spotted on microscopy glasses and stained with May-Grünwald Giemsa stain before analysis with a light microscope. Pictures were taken at 40× magnification and are representative of 6 different experiments. (D,E) Cells were double labeled for CXCR4 and CD16 and analyzed by flow cytometry. Data are expressed as a percentage of the maximum fluorescence for the control sample at 0 hours for CD16 and 24 hours for CXCR4. All graphs represent the means (± SEM) of 6 different experiments performed in duplicate.