Macrophage-derived monocytes selectively downregulate CCR2, but not CCR1, during differentiation. (a). Changes in morphology between freshly isolated monocytes (left panel) and cells cultured for 5 days (right panel) were determined using a Nikon Diaphot Camera set up and Axon Imaging Workbench software. Magnification is at 60 ×. (b). Freshly isolated monocytes were either cultured for 5 days (broken line) or immediately stained (solid line) for a panel of macrophage markers: CD36 (left panel), CD11b (middle panel) or CD68 (right panel). Dotted histograms represent the isotype controls. (c). Panel I. Genomic DNA was prepared from freshly isolated monocytes and assayed for germ line expression of chemokine receptors CCR1-CCR9 and CXCR1-CXCR5 by PCR using primers designed in-house. Note each primer pair amplified a single product only, thus confirming that the primers are functional and specific. Panel II. Messenger RNA was prepared from freshly isolated monocytes (upper panel) and cells that had been cultured for either 2 days (middle panel) or 5 days (lower panel). Subsequently, RT-PCR was performed using primers for chemokine receptors CCR1-CCR9, CXCR1-CXCR5 and GAPDH. Marker is a 100 bp DNA ladder. Similar results were obtained in three other experiments. (d). Freshly isolated monocytes (upper panel plots 1, 4, 7, 10, 13 and 16) and cells that had been cultured for either 2 days (middle panel plots 2, 5, 8, 11, 14 and 17) or 5 days (lower panel plots 3, 6, 9, 12, 15 and 18) were stained for CCR1, CCR2, CCR5, CCR7, CXCR2 and CXCR4. Cells were then analyzed for changes in chemokine receptor expression by flow cytometry. Similar results were obtained in three other experiments.