EX-VIVO PKH26-LABELING OF LYMPHOCYTES FOR STUDIES OF CELL-MIGRATION IN-VIVO

A prerequisite for studies of cell migration is that the cells of inte rest can be appropriately labelled and subsequently easily traced. The use of radioisotopes or fluorescent substances that bind covalently t o the cell surface, e.g. fluorescein isothiocyanate (FITC) or rhodamin e isothiocyanate (RITC), have limitations such as rapid loss of the la belling, toxicity and interference with cell surface molecules. In the present work the authors labelled rat spleen lymphocytes with the flu orescent labelling molecule PKH26, which is incorporated into the lipi d bilayer of cytoplasmic membranes. The labelled lymphocytes were inje cted intravenously into syngeneic recipients and 2 or 6 days later the lymphocytes were detected in various organs by using flow cytometry a nd fluorescence microscopy. As could be expected, the lymphocytes home d to lymphoid tissues, preferably the spleen, and no labelled cells we re found in non-lymphoid organs such as the heart and the kidney. Memb rane labelling proved to be intense, uniform and stable and PKH26 posi tive cells were easily detectable in fractions less than 0.2% in perip heral blood and the various tissues after 6 days of in vivo circulatio n. Thus, the PKH26 dye appears to be suitable for labelling cell popul ations used in the study of cell migration in vivo, both under normal conditions and when specific immunological processes are taking place, such as graft rejection and tumour growth.