Fig. 6. (A-D) Colocalisation of cathepsin X (green; Alexa-Fluor-488) and LFA-1 (red; Alexa-Fluor-633) in Jurkat T lymphocytes migrating on ICAM1 (A,B) and Matrigel (C,D). Cells were seeded on pre-coated slides and allowed to migrate for 15 minutes (ICAM1) or 48 hours (Matrigel) before labelling. (A) In wild-type Jurkat T lymphocytes no morphological changes were observed, cathepsin X and LFA-1 are colocalised mainly in the perimembranous region. (B) In cathepsin-X-overexpressing Jurkat T lymphocytes with a polarised migratory phenotype cathepsin X and LFA-1 are colocalised predominantly at the uropod. (C,D) A similar colocalisation profile was obtained in 3D Matrigel. In cathepsin-X-overexpressing cells (D) with a polarised migratory phenotype cathepsin X and LFA-1 are colocalised at the perimembrane region and at the uropod. Fluorescent dyes were imaged sequentially in all colocalisation experiments in a frame-interlace mode to eliminate cross talk between the channels. The threshold level for this display was set to 90, which corresponds to two-thirds of the maximal brightness level. Pixels above the threshold in both channels (blue to white colour) and the contour plot are shown for images that demonstrate colocalisation. Scale bars, 20 µm.