Intracellular distribution of FL in resting and activated T lymphocytes.

Two-color immunofluorescence confocal microscopy was performed with resting cells (ex vivo, A,B) and cells cultured with IL-7 (C,D), or IL-7 plus CsA (E,F). For culture conditions, see legend to Figure 5. Cells were stained with anti-FL mAb M5 followed by IgG-Cy2 and with Abs against giantin or TGN46 followed by IgG-TexasRed. Images A to F are merged maximum-intensity projections of 20 optical sections; an exact overlap of green FL signals with red signals of giantin or TGN46 is depicted in yellow. FACS (G) and 3-color immunofluorescence confocal microscopy (H) were performed with cells cultured with IL-2 (15 ng/mL) plus BfA (10 μg/mL; added for the last 16 hours of culture). For FACS analysis, mFL was stained with mAb M5 followed by IgG-FITC; mFL expression in IL-2–stimulated cells in the absence of BfA is shown. Dotted line indicates staining with isotype-matched control mAb. For confocal microscopy, cells were stained with anti-FL mAb M5 followed by IgG-Cy2, with antibody against calnexin followed by IgG-Cy3, and with mAb against giantin followed by IgG-Cy5. Colocalization of FL and giantin with calnexin is depicted in white, colocalization of FL with calnexin in yellow, and colocalization of calnexin with giantin in magenta. Image H represents a single section. A detector in the backchannel of the microscope was used for recording differential interference contrast images (A-F and H) of the cells.