Figure 1
Figure 1. Effects of G-CSF on the growth, differentiation, and CXCR4 expression in the IL-3–dependent 32Dcl3 myeloid cells. (A) DNA synthesis in 32Dcl3 cells cultured for 24 to 144 hours in the presence of IL-3 alone or IL-3 plus G-CSF measured by 3H thymidine incorporation. The error bars represent standard deviation (SD) of the mean (triplicate cultures). (B) Microscopic morphology of 32Dcl3 cells cultured for 72 and 144 hours with IL-3 alone or with IL-3 plus G-CSF. Cytospin preparations were stained with May-Grünwald-Giemsa and mounted with PBS/1% glycerol. Cells were visualized through an Eclipse 6600 Nikon microscope (Melville, NY) equipped with a 60×1.40 oil objective (Nikon), and photographed with a Cool Snaps fx Roper Camera (Roper Scientific, Duluth, GA). Images were acquired with IPlab software (Biovision Technologies, Fairfax, VA) and imported into Adobe Photoshop 7.0 software (Adobe, San Jose, CA). (C) Western blot analysis of CXCR4, MPO, and actin content in 32Dcl3 cells cultured for 24 to 144 hours with IL-3 alone or with IL-3 plus G-CSF. The blot was stained with antibodies to CXCR4 then stripped and stained for actin first and then for MPO. (D) Flow cytometric analysis of CXCR4 and Gr-1 surface levels in 32Dcl3 cells cultured with IL-3 alone or IL-3 plus G-CSF for 72 and 144 hours. Background fluorescence with control antibodies is also shown (dotted line). (E) Transwell migration of 32Dcl3 cells in response to recombinant SDF-1α or serum. Cells were precultured for 3 days with IL-3 alone or with IL-3 plus G-CSF. The results (expressed as the percentage of input cells) reflect the mean of 3 experiments, each performed in triplicate. The error bars represent SD of the mean (*P < .05). (F) Attachment of 32Dcl3 cells to wells coated with SDF-1α or medium only. Cells were precultured for 3 days with IL-3 alone or with IL-3 plus G-CSF. The results reflect the means of 3 experiments, each performed in 5 replicates. Results are expressed as the percentage of input cells; error bars represent SD of the mean (*P < .05).

Effects of G-CSF on the growth, differentiation, and CXCR4 expression in the IL-3–dependent 32Dcl3 myeloid cells. (A) DNA synthesis in 32Dcl3 cells cultured for 24 to 144 hours in the presence of IL-3 alone or IL-3 plus G-CSF measured by 3H thymidine incorporation. The error bars represent standard deviation (SD) of the mean (triplicate cultures). (B) Microscopic morphology of 32Dcl3 cells cultured for 72 and 144 hours with IL-3 alone or with IL-3 plus G-CSF. Cytospin preparations were stained with May-Grünwald-Giemsa and mounted with PBS/1% glycerol. Cells were visualized through an Eclipse 6600 Nikon microscope (Melville, NY) equipped with a 60×1.40 oil objective (Nikon), and photographed with a Cool Snaps fx Roper Camera (Roper Scientific, Duluth, GA). Images were acquired with IPlab software (Biovision Technologies, Fairfax, VA) and imported into Adobe Photoshop 7.0 software (Adobe, San Jose, CA). (C) Western blot analysis of CXCR4, MPO, and actin content in 32Dcl3 cells cultured for 24 to 144 hours with IL-3 alone or with IL-3 plus G-CSF. The blot was stained with antibodies to CXCR4 then stripped and stained for actin first and then for MPO. (D) Flow cytometric analysis of CXCR4 and Gr-1 surface levels in 32Dcl3 cells cultured with IL-3 alone or IL-3 plus G-CSF for 72 and 144 hours. Background fluorescence with control antibodies is also shown (dotted line). (E) Transwell migration of 32Dcl3 cells in response to recombinant SDF-1α or serum. Cells were precultured for 3 days with IL-3 alone or with IL-3 plus G-CSF. The results (expressed as the percentage of input cells) reflect the mean of 3 experiments, each performed in triplicate. The error bars represent SD of the mean (*P < .05). (F) Attachment of 32Dcl3 cells to wells coated with SDF-1α or medium only. Cells were precultured for 3 days with IL-3 alone or with IL-3 plus G-CSF. The results reflect the means of 3 experiments, each performed in 5 replicates. Results are expressed as the percentage of input cells; error bars represent SD of the mean (*P < .05).

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