**6. Summary**

To explore the CML-derived extracellular vesicles impact on T regulatory cell development and

**Figure 8.** Flow cytometry analysis of T regulatory (Treg) cells differentiation and function. (A) Analysis of cell viability and proliferation (AmCyan channel—fixable viability stain eFluor 506, V450—violet proliferation dye 450). V450 staining allows to track thymocyte proliferation and distinguish them from JAWS II cells (V450-). (B) Identification of T regulatory cells by sequential gating and analysis of CTLA-4 expression. (C) CFSE staining allows to track proliferation of responder cells in an *in vitro* suppression test. An example of combination with CD4-APC staining, which helps

To study the effect of CML extracellular vesicles on the differentiation of thymic regulatory T cells (tTreg), we have employed a previously described *in vitro* assay of tTreg differentiation on JAWS II immature dendritic cells [80]. Briefly, during the step of preactivation of thymocytes on αCD3-coated plates, we added extracellular vesicles to allow their uptake or binding solely by thymocytes. Thymocytes were additionally stained with a violet proliferation dye 450, which would allow to track their proliferation and distinguish them from JAWS II cells. After 24 h of preactivation, thymocytes were washed and co-cultured with JAWS II cells. Afterwards, tTreg differentiation using multicolor flow cytometry was analyzed. This analysis allowed to track viability, proliferation (**Figure 8A**), differentiation of tTreg, as well as their phenotype (**Figure 8B**). Extracellular vesicles can also potentially influence suppressive activity of mature, already differentiated tTreg. To study this phenomenon, we sorted tTreg and cultured them, first with CML extracellular vesicles for 24 h and afterwards with conventional T cells (responder cells) stimulated for proliferation, to observe differences in suppressive activity manifested by decreased

**5.1. Flow cytometry analysis of T regulatory (Treg) cells differentiation and function**

activity, we adjust two *in vitro* assays, both based on the flow cytometry.

eliminate remaining Treg cells (CFSE-, CD4+) in analysis, is presented.

34 Multidimensional Flow Cytometry Techniques for Novel Highly Informative Assays

Many studies already confirmed that leukemia cells behave differently whether they are cultured alone or in co-culture with stromal cells. This mimics some elements of the bone marrow microenvironment and represents more physiological conditions. Also, the cross talk between leukemia and immune cells is an example showing importance of the interactions within the leukemia microenvironment. Thus, in our opinion, analysis of cell signaling in the co-culture conditions is highly informative and might have some therapeutic implications.

Studies of leukemia cells *in vitro/ex vivo*, including analysis of primary cells and stem/progenitor subpopulations, represent an important step in development of novel therapeutic strategies. Different parameters, such as viability, proliferation, DNA damage, clonogenic potential, and others, are investigated after treatment with drugs or drug candidates. Pretreatment with investigated drugs can also be followed by mice Xenograft *in vivo* studies to investigate their therapeutic potential. All these studies are critically important as a first step in analysis of potential therapeutic strategies.

Nevertheless, we propose that in the next step or simultaneously, some of these studies might be performed also upon co-culture conditions to add an essential element of the cross-talk with stroma components, which normally exist *in vivo*. These conditions allow to verify whether the treatment is still effective even upon protective impact of stroma. This fulfills, at least partially, the big gap between *in vitro* experiments and *in vivo* conditions.

In this chapter, we described and discussed flow cytometry applications, which can be used to perform co-culture studies to analyze some signaling elements within the leukemia microenvironment. This strategy enables to distinguish between the cell types and to investigate the cross talk between cancer and surrounding cells, signaling pathways regulated by the cell-cell interactions, as well as sensitivity to treatment with anticancer drugs in the stroma-mimicking conditions. Utilizing modern flow cytometry and a broad spectrum of currently available dyes/trackers allows to perform highly informative studies not only because of the use of multiparameter cytometry but also because of more complex cellular context, which is taken under consideration. Moreover, even if the described flow cytometry applications rely on the leukemia microenvironment studies, they are uniform and can be broadly applied into another biological context.
