Optimization of 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester for labeling human intervertebral disc cells in vitro

We have assessed the utility of an intracellular fluorochrome, 5-(and-6)-ca rboxyfluorescein diacetate succinimidyl ester (CFSE), as a tracking label f or human intervertebral disc cells in vitro. Although 5 mu M provides adequ ate intracellular labeling for whole cell fluorescent microscopic identific ation of labeled cells, 20 mu M was preferable for immunocytochemical local ization of paraffin embedded labeled cells. Electron dense vesicles are see n at the ultrastructural level in labeled cells. Discrete vesicular labelin g can also be observed in whole cell mounts viewed with fluorescence micros copy. Whole cells retain good label for 6 weeks. CFSE labeling is relativel y easy, nontoxic to cells and nonradiocactive. Initial optimization of clos e with specific cells types is recommended when confirmation of positive im munocytochemistry is needed for tissue engineering studies.