Long-term tracking of lymphocytes in vivo: the migration of PKH-labeled lymphocytes

Studies of in vivo cell migration using cell markers such as 51Cr, 111In, FITC, or XRITC have been limited to short time periods due to the elution, toxicity, or rapid loss of label detectability. We have labeled sheep lymphocytes in vitro with PKH-2, a new fluorescent cell membrane label, and, after their intravenous injection back into donor sheep, have been able to detect them in efferent lymph, using flow cytometry, for longer than 38 days. The PKH-2-labeled lymphocytes migrated with similar kinetics, efficiency, and tissue specificity as lymphocytes labeled with cell markers used previously. PKH-2-labeled cells mediated graft versus host reactions indistinguishable from those mediated by unlabeled cells, and cell surface antigens were equally detectable on the surface of labeled and unlabeled lymphocytes. According to the slow, consistent loss of fluorescence intensity of the labeled cells in vivo, we predict that labeled lymphocytes could remain detectable by flow cytometry for greater than 7 weeks with the labeling protocol used in these experiments.