After harvesting, the cells may be labeled as desired and according to standard protocol. For the acquisition and analysis of data, cells could be looked at firstly in a FSC/SSC plot and a gate set around the cells of interest. Depending on the experiment setup, it may be useful to include a dead cell exclusion dye. The viable gate can then be used in a dot plot showing CFDA, SE versus, for example, side scatter or CD3. For the beginner, it may at this point be desirable to have quite a large number of positive control cells so that adequate time can be spent to find the different peaks and to establish the relevant compensation settings. If a large number of cells have proliferated, it may not be completely straightforward to find the typical gradual decrease in fluorescence that is so often shown in CFDA, SE plots. Adjusting the setting to view only 20-50% or so of the total cell number could be helpful in these cases. Also, if cells have been treated with a powerful antigen for 7 d and the original culture contained only T cells, or even a primary antigen-specific T-cell line, it is not unlikely that all cells have proliferated at least once. Thus, there may not be any "zero cycle" point present if only CD3+ or CD4+ T cells are assessed. The zero cycle point is perhaps not always of interest for the researcher; however, a zero cycle of proliferation is required if the precursor frequency is to be estimated (see Subheading 4.7. for precursor frequency calculation). Here, the problem could be solved by including the antigen-presenting cell population; these cells do not proliferate and therefore can serve to represent the original, zero cycle peak. As always in flow cytometry and labeling of cells with different sizes and intracellular content, larger cells such as monocytes or Epstein-Barr-transformed B-cell lines will have higher CFDA, SE fluorescence intensity than the smaller T cells. Thus, the first cell proliferation cycle after a monocyte-indicated zero cycle point may not occur at exactly half the fluorescence intensity of the monocyte peak but rather a bit further toward the intercept. Another option is to use the negative control (that is, tested cells and antigen-presenting cells cultured without antigen). For the beginner, it could be helpful to combine a few cells from the nonantigen and the antigen culture in one tube, to very easily compare the peaks between these two T-cell populations.
It is difficult to say where exactly the detection limit of proliferation occurs. With regard to cycles of division, eight to 10 successive generations of lymphocytes have been demonstrated to be resolved (17,18). There could also be limits with regard to how many total events are available if cell cultures of quite precious material are studied, and so even if 1-5% of the total survived population are cells that have proliferated, it may prove difficult to record a high enough number of total events to reach a good cluster of proliferated events. As previously mentioned in the rare event analysis section, if only a few events can be identified, the CV will be high and ultimately it is up to the researcher to determine what an acceptable CV is. Figure 3 shows an example of a relatively minor population of proliferating T cells. The experiment was part of a study investigating autoantigen-induced proliferation of CD4+ T cells, and a minimum of 100 events were collected in the proliferation-positive CD4+ gate, giving a CV of 10%. If a small population is followed, it is recommended to use the appropriate controls as outlined earlier and, whenever possible, to repeat the experiments. The section on rare event analysis deals further with some of the issues related to the analysis of small populations.
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