Localization and expression of STAT3 and CD15 proteins during ex vivo granulocytic differentiation.

CD34⁺ cells from mobilized peripheral blood were directed along the granulocyte lineage with SCF, IL-6, G-CSF, with or without IL-3, and harvested for analysis by dual-color immunofluorescence microscopy. Detection of STAT3 and CD15 in cultures at (A) 5% O₂, pH 7.25, +IL-3 (control condition); (B) 5% O₂, pH 7.4, +IL-3 (high pH); and (C) 5% O₂, pH 7.25, −IL-3. Negative controls (background) for each day of analysis using FITC- and Texas Red–conjugated secondary antibodies in the absence of the relevant primary antibodies are shown in the first row. The same field of cells is shown in the upper and lower panels of panels A-C. Upper panels of panels A-C: DAPI staining of nucleic acid (white). Lower panels of panels A-C: codetection of STAT3 (green) and CD15 (red) using polyclonal rabbit anti-STAT3 and monoclonal mouse anti-CD15 primary antibodies, followed by FITC-conjugated goat antirabbit IgG and Texas Red–conjugated goat antimouse IgM μ-chain–specific secondary antibodies. Images were captured using a × 40 oil immersion objective and are representative of 3 randomly selected fields per culture condition for each of 3 different sets of experiments. No appreciable differences in STAT3 and CD15 staining were visible in cells cultured at 20% O₂ versus the control condition (data not shown). A 10-μm scale bar is shown in the day 0 panels.