Background Information

Both the hematopoietic and immune systems are characterized by a requirement for a large amount of cell division, coupled with differentiation to specialized effector cells. As these systems consist predominantly of free cells, the study of their lineage relationships, cellular division, and differentiation has been difficult. Crude, bulk measures of division and differentiation do not allow the subtleties of such complex systems to be explored.

The intracellular dye CFSE is cell permeant and remains nonfluorescent until cellular esterases cleave carboxyl groups from the molecule, rendering it both nonpermeant and fluorescent. In addition, the succinimidyl moiety covalently attaches to amine groups, and thereby contributes to the longevity of staining. During cell division, CFSE is distributed equally between daughter cells. This has been validated by simultaneous quenching of Hoechst dye fluorescence by BrdU to determine cell division (Lyons and Parish, 1994). Depending on the intensity of the starting fluorescence, around eight discrete division cycles can be determined using this standard protocol in hematopoietic cells. The number of divisions which can be followed is limited only by the autofluorescence level of unlabeled cells and the uniformity in size of the labeled cell population. As a dividing cell population approaches this autofluorescence level, the division peaks start to compress, preventing resolution beyond about eight cycles (Hodgkin et al., 1996).

The standard protocol is used for tracking division over a period of 2 to 14 days, and is suitable for most in vitro applications, as well as for in vivo transfers over a similar time frame. On a flow cytometer set up for detecting a bright fluorescein-conjugated antibody, undivided cells will have a fluorescence intensity in the fourth decade on a logarithmic scale.

For longer-term tracking, more intense staining may be required. The optimal starting fluorescence intensity will depend primarily on the length of time cells will be tracked and on the inherent resilience of the population of choice to the toxicity associated with intense CFSE staining. In the first day or two after staining, fluorescence intensity drops by about 60% in the absence of division (Fig. 9.11.1), as the more labile constituents binding CFSE are catabolized; thereafter, the intensity remains relatively stable for several weeks to months. This initial decrease does not affect the ability to follow division, as such losses are proportional regardless of cell division. The starting intensity of fluorescence obtained is essentially linear with respect to CFSE concentration (Figure 9.11.2) and duration of incubation with CFSE; therefore, the level of staining may be manipulated to suit the experiment.

The CFSE method has been successfully used to determine division-related rules of im-munoglobulin isotype switching by murine B lymphocytes (Hodgkin et al., 1996; Hasbold et al., 1998) and in vitro proliferative behavior of T lymphocytes (Wells et al., 1997; Fig. 9.11.3), B lymphocytes (Kindler and Zubler, 1997), and human hematopoietic precursors (Nordon et al., 1997). It can be used to model in vivo phenomena, such as the alloresponse to major histocompatibility complex mismatch (Fig. 9.11.5; Table 9.11.1). The technique has also

Studies of Cell Function

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