IL-3 and pH modulate STAT3 and CD15 expression as well as changes in nuclear morphology during granulocytic differentiation.
Similar to Figure 1, except that immunofluorescence was evaluated on days 3, 7, and 15 of culture for images captured using a × 100 oil immersion objective. Images are representative of 2 randomly selected fields per culture condition for each of 3 different sets of experiments. The same field of cells is shown in the upper and lower panels of each day for each culture condition. Left panels: negative control (background) using FITC- and Texas Red–conjugated secondary antibodies in the absence of the relevant primary antibodies. Upper panels of each day: DAPI staining of nucleic acid (white). Lower panels of each day: codetection of STAT3 (green) and CD15 (red) using polyclonal rabbit anti-STAT3 and monoclonal mouse anti-CD15 primary antibodies, followed by FITC-conjugated goat anti–rabbit IgG and Texas Red–conjugated goat anti–mouse IgM μ-chain–specific secondary antibodies. DAPI staining indicates that cells sequentially exhibit features characteristic of typical granulocytic maturation—from the enlarged nucleus of the immature myeloblast to the lobulated nucleus of the mature neutrophil—with their progression dependent on the culture conditions. Arrows in day 3 panels indicate nucleoli. Arrows in day 7 panels indicate the decreased nuclear-cytoplasmic ratio of the myelocyte stage. Arrows in day 15 panels indicate typical nuclear morphology of metamyeloctyes (M), bands (B), and segmented neutrophils (SN). Advancement through the morphologic transitions did not appear different at 20% O2 compared with control cultures (data not shown). Ten-micrometer scale bars are shown in the negative control panels.
