TY - JOUR
T1 - Effects of fluorescent and nonfluorescent tracing methods on lymphocyte migration in vivo
AU - Nolte, Martijn A
AU - Kraal, Georg
AU - Mebius, Reina E
N1 - Copyright 2004 Wiley-Liss, Inc
PY - 2004/9
Y1 - 2004/9
N2 - BACKGROUND: The use of fluorescent dyes to monitor in vivo cellular migration and proliferation has greatly expanded, but little is known about their potential influence on cell migration.METHODS: Adoptive transfer studies of lymphocytes labeled with various dyes were performed, and their in vivo homing was compared with that of coinjected unlabeled control cells. In addition, in vitro migration and binding studies were performed to analyze the various steps of transmigration separately.RESULTS: These data showed that the intracellular fluorescent dyes calcein acetoxymethyl ester, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester, 5-chloromethylfluorescein diacetate, 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester, and fluorescein isothiocyanate affect in vivo homing of especially B lymphocytes to lymphoid organs, without any direct effect on in vitro chemotactic or adhesive activity. The only label that did not affect migration was the extracellular and nonfluorescent molecule biotin, provided that the labeling was performed at room temperature. Interestingly, by using the highly versatile congenic Ly5.1-Ly5.2 system, we also demonstrated intrinsic differences in lymphocyte migration based on allelic differences.CONCLUSIONS: Our data showed that fluorescent labeling of lymphocytes has a severe effect on their homing capacity in vivo. Labeling of cells with biotin appeared to be a good alternative for this purpose; however, if direct fluorescence is required, the negative effects on cell migration should be considered.
AB - BACKGROUND: The use of fluorescent dyes to monitor in vivo cellular migration and proliferation has greatly expanded, but little is known about their potential influence on cell migration.METHODS: Adoptive transfer studies of lymphocytes labeled with various dyes were performed, and their in vivo homing was compared with that of coinjected unlabeled control cells. In addition, in vitro migration and binding studies were performed to analyze the various steps of transmigration separately.RESULTS: These data showed that the intracellular fluorescent dyes calcein acetoxymethyl ester, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester, 5-chloromethylfluorescein diacetate, 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester, and fluorescein isothiocyanate affect in vivo homing of especially B lymphocytes to lymphoid organs, without any direct effect on in vitro chemotactic or adhesive activity. The only label that did not affect migration was the extracellular and nonfluorescent molecule biotin, provided that the labeling was performed at room temperature. Interestingly, by using the highly versatile congenic Ly5.1-Ly5.2 system, we also demonstrated intrinsic differences in lymphocyte migration based on allelic differences.CONCLUSIONS: Our data showed that fluorescent labeling of lymphocytes has a severe effect on their homing capacity in vivo. Labeling of cells with biotin appeared to be a good alternative for this purpose; however, if direct fluorescence is required, the negative effects on cell migration should be considered.
KW - Animals
KW - Biotin/metabolism
KW - Cell Movement/drug effects
KW - Cell Transplantation
KW - Fluorescein-5-isothiocyanate/chemistry
KW - Fluorescent Dyes/chemistry
KW - Lymphocytes/cytology
KW - Mice
KW - Mice, Inbred C57BL
KW - Molecular Structure
U2 - 10.1002/cyto.a.20074
DO - 10.1002/cyto.a.20074
M3 - Article
C2 - 15351987
VL - 61
SP - 35
EP - 44
JO - Cytometry Part B. Clinical Cytometry
JF - Cytometry Part B. Clinical Cytometry
SN - 1552-4949
IS - 1
ER -