Effects of fluorescent and nonfluorescent tracing methods on lymphocyte migration in vivo
Article first published online: 6 AUG 2004
Copyright © 2004 Wiley-Liss, Inc.
Cytometry Part A
Volume 61A, Issue 1, pages 35–44, September 2004
How to Cite
Nolte, M. A., Kraal, G. and Mebius, R. E. (2004), Effects of fluorescent and nonfluorescent tracing methods on lymphocyte migration in vivo. Cytometry, 61A: 35–44. doi: 10.1002/cyto.a.20074
- Issue published online: 17 AUG 2004
- Article first published online: 6 AUG 2004
- Manuscript Accepted: 18 MAY 2004
- Manuscript Revised: 12 APR 2004
- Manuscript Received: 17 DEC 2003
- lymph node;
- 5-(and-6)-carboxyfluorescein diacetate;
- succinimidyl ester;
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.
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.
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.
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. © 2004 Wiley-Liss, Inc.