**3. Fluorescent cell tracking to follow cell type, function, and state in co-cultures**

Upon co-culture conditions, when different types of cells are cultured together, the key component of the experimental strategy enabling cell analysis is to separate both cell types, either physically by sorting or by flow cytometry gating. For this, cells have to express some fluorescent protein or have to be stained and tracked by fluorescent dye. This allows to separate signals from each type of cell based on the different fluorochromes using flow cytometry. The scheme and typical histograms to analyze tracked cells in co-culture are presented in **Figure 1**.

**Function Excitation laser [nm] Dye Emission (nm)**

488 (blue) CFSE 520

530 (yellow-green) CellTrace yellow cell proliferation kit 579 560 (yellow) CellTrace yellow cell proliferation kit 579 633/640 (red) CellTrace far red cell proliferation kit 661

405 (violet) CellTrace calcein violet 452

488 (blue) CellTrace calcein green 515

530 (yellow-green) SYTOX orange dead cell stain 570

560 (yellow) Propidium iodide 617

633/640 (red) TO-PRO™-3 iodide 661

405 (violet) Live/dead fixable dead cell stain kit (450) 450

488 (blue) Fixable viability stain 520 520

Fixable viability 355 (UV) Live/dead fixable dead cell stain kit (450) 450

Violet proliferation dye 450 450

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EdU proliferation kit (iFluor 488) 520

Hoechst 33258 450 DAPI 470

SYTOX blue dead cell stain 480

SYTOX green dead cell stain 523 TO-PRO™-1 iodide 530 SYTOX orange dead cell stain 570 Propidium iodide 617 7-AAD (7-aminoactinomycin D) 647

Propidium iodide 617 7-AAD (7-aminoactinomycin D) 647

7-AAD (7-aminoactinomycin D) 647

Fixable viability stain 450 450 Fixable viability stain 510 510 Live/dead fixable dead cell stain kit (525) 525 Live/dead fixable dead cell stain kit (575) 575 Fixable viability stain 575 V 575

Live/dead fixable dead cell stain kit (530) 530 Live/dead fixable dead cell stain kit (585) 585

Proliferation 405 (violet) CellTrace violet cell proliferation kit 450

Viability 355 (UV) CellTrace calcein blue 425

**Figure 1.** Scheme of the cell type separation by fluorescent tracking followed by flow cytometry gating.

#### **3.1. Fluorescent cell trackers**

Cell trackers have been successfully used for years in multiple studies, starting from immunology, which intensively utilized flow cytometry techniques [17–19]. Today, there is a broad spectrum of commercially available tracking dyes, which simultaneously allow to track cells and study proliferation and/or viability. Cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. They are represented by "membrane dyes," which are highly lipophilic dyes that go stably but noncovalently into cell membranes or "protein dyes," which are amino-reactive dyes that form stable covalent bonds with cell proteins [20]. They are safe and can be used in viable cells even in the long-term cultures. Big variability allows excitation by any available laser and usage in combination with other fluorochromes in multiparameter studies. **Table 1** presents the tracking dyes currently available for different lasers. These dyes can be used separately or combined due to their different excitation signals. It is important to mention that due to its nature, the fluorescent signal of labeled population is decreasing, depending on the growth rate of cells. So, quickly dividing cells will lose tracing signal faster than cells with longer cell cycle. This feature, on the one hand, enables monitoring of cell proliferation rate in co-culture setups but on the other hand limits the time frame within which the subpopulation of dye-labeled cells can be distinguished. **Figure 2** presents an example of the time-course of CMAC fluorescence measured by flow cytometry, when different concentrations of CMAC dye were used. After 72 h, two populations were not clearly separated anymore when the lower concentration of CMAC was used. Therefore, also concentration of the dye used for labeling of different types of cells should be chosen accordingly to the level of fluorescence detected at the end time point of planned experiment and verified in the toxicity assay.

Another strategy to track cells is expression of fluorescent proteins. Green fluorescent protein (GFP) has been purified in the 60-ties from *Aequorea victoria* [21]. This has started a new era of studies, in which original or engineered GFP as well as other fluorescent proteins were utilized to tag proteins of interest. Today, GFP derivatives, yellow fluorescent protein (YFP) and a big family of red fluorescence proteins (RFP), which are derivatives of DsRed isolated from *Discosoma* sp. [22], are commonly used in many different studies. The advantage of this strategy is stable fluorescent signal; however, the stable expression of fluorescent proteins has some limitations, requires transfection of cells and is connected with the nonphysiological Insight into the Leukemia Microenvironment and Cell-cell Interactions Using Flow Cytometry http://dx.doi.org/10.5772/intechopen.76481 23


**3.1. Fluorescent cell trackers**

Cell trackers have been successfully used for years in multiple studies, starting from immunology, which intensively utilized flow cytometry techniques [17–19]. Today, there is a broad spectrum of commercially available tracking dyes, which simultaneously allow to track cells and study proliferation and/or viability. Cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. They are represented by "membrane dyes," which are highly lipophilic dyes that go stably but noncovalently into cell membranes or "protein dyes," which are amino-reactive dyes that form stable covalent bonds with cell proteins [20]. They are safe and can be used in viable cells even in the long-term cultures. Big variability allows excitation by any available laser and usage in combination with other fluorochromes in multiparameter studies. **Table 1** presents the tracking dyes currently available for different lasers. These dyes can be used separately or combined due to their different excitation signals. It is important to mention that due to its nature, the fluorescent signal of labeled population is decreasing, depending on the growth rate of cells. So, quickly dividing cells will lose tracing signal faster than cells with longer cell cycle. This feature, on the one hand, enables monitoring of cell proliferation rate in co-culture setups but on the other hand limits the time frame within which the subpopulation of dye-labeled cells can be distinguished. **Figure 2** presents an example of the time-course of CMAC fluorescence measured by flow cytometry, when different concentrations of CMAC dye were used. After 72 h, two populations were not clearly separated anymore when the lower concentration of CMAC was used. Therefore, also concentration of the dye used for labeling of different types of cells should be chosen accordingly to the level of fluorescence detected at the end time point of planned experiment and verified in the toxicity assay. Another strategy to track cells is expression of fluorescent proteins. Green fluorescent protein (GFP) has been purified in the 60-ties from *Aequorea victoria* [21]. This has started a new era of studies, in which original or engineered GFP as well as other fluorescent proteins were utilized to tag proteins of interest. Today, GFP derivatives, yellow fluorescent protein (YFP) and a big family of red fluorescence proteins (RFP), which are derivatives of DsRed isolated from *Discosoma* sp. [22], are commonly used in many different studies. The advantage of this strategy is stable fluorescent signal; however, the stable expression of fluorescent proteins has some limitations, requires transfection of cells and is connected with the nonphysiological

**Figure 1.** Scheme of the cell type separation by fluorescent tracking followed by flow cytometry gating.

22 Multidimensional Flow Cytometry Techniques for Novel Highly Informative Assays


which can be utilized, such as CBD500, Pacific Orange NHS ester, DyLight 350 NHS ester, and DyLight 800 NHS ester, Pacific Blue dyes, Alexa dyes, eFluor, and others. Using this technique allows to perform multiplate assays in relatively short time and with economical use of reagents, as mixing sample prior to staining, antibody consumption is reduced 10- to 100-fold. Importantly, either viable or fixed samples can be barcoded, thus giving broad possibilities to combine FCB with other multiparameter protocols. Such strategy can be used to analyze samples under treatments, co-cultured, or stimulated with different agents. FCB has been successfully utilized for different studies, including drug screening and cell signaling profiling studies [26], intracellular cytokine production [27], immunophenotyping [28], and others.

**Figure 2.** The time-course of CMAC fluorescence measured by flow cytometry upon conditions when different concentrations (10 and 25 μM) of CMAC dye were used. CMAC-positive (CMAC+, blue) subpopulation is gated. Green

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Quantitative assessment of proliferation is an important parameter in leukemia and other cancer studies. Especially, when co-culture with stroma provides protective signaling, proliferation is analyzed as one of the major parameters to estimate cytostatic effects. Moreover, proliferation dyes can also be utilized to fluorescently track cells in co-culture. So, in addition to detection and quantifying cell divisions, these dyes can be used to distinguish one type of cells from the other. The measurement, independently on the tracking dye used, relies on the same mechanism, namely permanent labeling of cells without affecting morphology or physiology to trace generations or divisions *in vivo* or *in vitro*. Dilutions of the dye equally distributed to daughter cells as a result of cell divisions can be measured by flow cytometry [17, 20]. The scheme of CFSE dilution with each cell division and flow cytometry measurement is presented in **Figure 3**.

For many years, CFSE dye (carboxyfluorescein succinimidyl ester) was a gold standard for tracking cell divisions [29]; however, today there is a variety of tracking dyes allowing to measure the proliferation index. Proliferation dyes are available for the UV, violet, blue, yellow, and red lasers, giving more flexibility in multiplexed experiments [29, 30] (see **Table 1**, Proliferation). Compared to the classical CFSE, modern proliferation trackers are less cytotoxic

**3.2. Cell proliferation dyes**

subpopulation represents CMAC-negative cells in co-culture.

**Table 1.** Tracking dyes used for fluorescent cell staining; function, excitation, and emission spectra as well as full name are shown.

overexpression of novel form of protein. On the other hand, the signal from the expressed fluorescent protein is relatively stable over time. Thus, this attitude might have advantage over cell tracking in case of long-term experiments.

Recently, a highly advanced methodology based on the lentiviral fluorescent genetic barcoding system for flow cytometry-based multiplex cell tracking has been used to follow clonal subpopulations in the real time [23]. Cells transduced with the lentiviral vectors are individually marked by a highly characteristic pattern of insertion sites inherited by all their progeny. The system allows creation of 26, 14, or 6 unique labels, and the color-coded populations can be tracked for up to 28 days.

Another strategy to perform high-content multiplex analysis is based on the fluorescent cell barcoding (FCB) technique [24, 25]. In FCB, each sample is labeled with a unique fluorescent signature (barcode), of different fluorescence intensity (due to dilutions) and/or emission wavelength, mixed together with other samples, then stained with antibodies or probes and analyzed by flow cytometry as a single sample. Today, there is a variability of FCB dyes Insight into the Leukemia Microenvironment and Cell-cell Interactions Using Flow Cytometry http://dx.doi.org/10.5772/intechopen.76481 25

**Figure 2.** The time-course of CMAC fluorescence measured by flow cytometry upon conditions when different concentrations (10 and 25 μM) of CMAC dye were used. CMAC-positive (CMAC+, blue) subpopulation is gated. Green subpopulation represents CMAC-negative cells in co-culture.

which can be utilized, such as CBD500, Pacific Orange NHS ester, DyLight 350 NHS ester, and DyLight 800 NHS ester, Pacific Blue dyes, Alexa dyes, eFluor, and others. Using this technique allows to perform multiplate assays in relatively short time and with economical use of reagents, as mixing sample prior to staining, antibody consumption is reduced 10- to 100-fold. Importantly, either viable or fixed samples can be barcoded, thus giving broad possibilities to combine FCB with other multiparameter protocols. Such strategy can be used to analyze samples under treatments, co-cultured, or stimulated with different agents. FCB has been successfully utilized for different studies, including drug screening and cell signaling profiling studies [26], intracellular cytokine production [27], immunophenotyping [28], and others.

#### **3.2. Cell proliferation dyes**

overexpression of novel form of protein. On the other hand, the signal from the expressed fluorescent protein is relatively stable over time. Thus, this attitude might have advantage

**Table 1.** Tracking dyes used for fluorescent cell staining; function, excitation, and emission spectra as well as full name

**Function Excitation laser [nm] Dye Emission (nm)**

24 Multidimensional Flow Cytometry Techniques for Novel Highly Informative Assays

Cell tracking 355 (UV) Hoechst 33342 450

530 (yellow-green) Fixable viability stain 620 620 560 (yellow) Fixable viability stain 570 570 633/640 (red) Fixable viability stain 660 660

405 (violet) CellTracker™ violet BMQC 516

488 (blue) CellTracker™ green CMFDA 517 530 (yellow-green) CytoPainter orange cell tracking staining kit 560 560 (yellow) CellTracker™ CM-DiI 570

633/640 (red) CellTracker™ deep red 650

Live/dead fixable dead cell stain kit (660) 660 Fixable viability stain 700 700 Live/dead fixable dead cell stain kit (780) 780

CytoPainter blue cell tracking staining kit 455 CellTracker™ blue CMF2HC 464 CellTracker™ blue CMAC 466

CytoPainter green cell tracking staining kit 520

CellTracker™ orange CMRA 575 CytoPainter red cell tracking staining kit 600 CellTracker™ CMTPX 602

CytoPainter deep red cell tracking staining kit 650

Recently, a highly advanced methodology based on the lentiviral fluorescent genetic barcoding system for flow cytometry-based multiplex cell tracking has been used to follow clonal subpopulations in the real time [23]. Cells transduced with the lentiviral vectors are individually marked by a highly characteristic pattern of insertion sites inherited by all their progeny. The system allows creation of 26, 14, or 6 unique labels, and the color-coded populations can

Another strategy to perform high-content multiplex analysis is based on the fluorescent cell barcoding (FCB) technique [24, 25]. In FCB, each sample is labeled with a unique fluorescent signature (barcode), of different fluorescence intensity (due to dilutions) and/or emission wavelength, mixed together with other samples, then stained with antibodies or probes and analyzed by flow cytometry as a single sample. Today, there is a variability of FCB dyes

over cell tracking in case of long-term experiments.

be tracked for up to 28 days.

are shown.

Quantitative assessment of proliferation is an important parameter in leukemia and other cancer studies. Especially, when co-culture with stroma provides protective signaling, proliferation is analyzed as one of the major parameters to estimate cytostatic effects. Moreover, proliferation dyes can also be utilized to fluorescently track cells in co-culture. So, in addition to detection and quantifying cell divisions, these dyes can be used to distinguish one type of cells from the other. The measurement, independently on the tracking dye used, relies on the same mechanism, namely permanent labeling of cells without affecting morphology or physiology to trace generations or divisions *in vivo* or *in vitro*. Dilutions of the dye equally distributed to daughter cells as a result of cell divisions can be measured by flow cytometry [17, 20]. The scheme of CFSE dilution with each cell division and flow cytometry measurement is presented in **Figure 3**.

For many years, CFSE dye (carboxyfluorescein succinimidyl ester) was a gold standard for tracking cell divisions [29]; however, today there is a variety of tracking dyes allowing to measure the proliferation index. Proliferation dyes are available for the UV, violet, blue, yellow, and red lasers, giving more flexibility in multiplexed experiments [29, 30] (see **Table 1**, Proliferation). Compared to the classical CFSE, modern proliferation trackers are less cytotoxic

**Figure 3.** The scheme of tracking cells with proliferation dye. Dilution of CFSE with cell divisions (left panel) and CFSE detection by flow cytometry (right panel) are shown.

and more stable. Importantly, what has been already mentioned above, decrease of the fluorescence signal resulting from dye dilution with every division has to be considered when tracking cells with proliferation dyes.

In our studies, in which GFP-expressing CML cells have been co-cultured with stroma fibroblasts labeled with CMAC dye, tracking of subpopulations has been combined with analysis of proliferation (**Figure 4**). To do so, subpopulations have been gated based on the fluorescent signal of the cell tracker, either GFP (A) or CMAC (B), followed by separate analysis of cell divisions in each subpopulation. Such strategy allowed to investigate the impact of co-culture conditions on the proliferation index of either leukemia or stromal cells. This protocols can also be utilized in co-cultures under treatment with anticancer drugs to verify the protective effect of stroma. Similar strategy has been used to investigate interactions of lymphocytes with mesenchymal stem cells [31] as well as in the co-cultures of healthy or malign breast tissue-derived stromal cells with human breast adenocarcinoma cell line MCF-7 [32]. Altogether, proliferation dyes represent a very potent group of tracking dyes analyzed with a broad range of available lasers and cytometers.

to suboptimal tracking combinations, reproducible labeling, combining of cell tracking dyes with fluorescent antibodies staining and others. Briefly, the recommendations include evaluating the dye's spectral profile and available cytometers/lasers to optimize compatibility with other fluorochromes and to minimize compensation problems, evaluating the effect of labeling on cell growth rate, testing cell divisions and dye dilutions to determine the maximum number of generations to be included when using dye dilution profiles, and

**Figure 4.** Analysis of cell proliferation estimated upon tracking with CMAC, combined with cell-type gating in co-culture of leukemia and stromal cells. (A) Leukemia cells expressed GFP protein (GFP+) and (B) stromal cells were tracked with CMAC (CMAC+). Labeling with CMAC allowed for cell-type gating and simultaneous analysis of cell divisions. CMAC

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As proliferation is tracked in living cells, it enables to combine analysis of cell proliferation with other live-cell applications, such as immunophenotyping, cell sorting, cell cycle analysis, mitochondrial potential, ROS studies, and others. This possibility has been utilized, for example, in the studies in which myelin-phagocytosing macrophages were co-cultured with CFSE-labeled T lymphocytes followed by analysis of surface receptors and role of nitric oxide [35]. In other studies, cell tracker has been used to investigate immune cells proliferation and

Another group of trackers is represented by the fixable viability dyes, enabling the post-fixation discrimination of dead cells, which lost plasma membrane integrity before fixation (see **Table 1**, Viability). This gives a novel opportunity to combine them with staining procedures which require fixation. Thus, analysis of internal targets, like as intracellular protein levels or γH2AX, within the population of cells which was alive prior fixation, was possible. In our

verifying that relevant cell functions remain unaltered by tracking dye labeling.

was detected in the APC channel; histograms show CMAC dilution at different time points of co-culture.

cytotoxicity [36].

#### **3.3. Multiparameter studies**

Modern flow cytometry, due to development of multiple laser systems, provides possibility for polychromatic analysis of many parameters in single cells. The key to success, particularly in the studies, in which multiple dyes are used to track different types of cell, is therefore to understand the critical issues enabling optimal use of different tracking dyes. It has to be noticed that some limitations appear when using tracking dyes in co-culture experiments. Each class of commercially available cell trackers—lipophilic "membrane" dyes and amino-reactive "protein" dyes possess advantages and limitations. Thus, there is a need for optimization of different proliferation tracking dyes especially when combined together [33]. Recently, a novel edition of protocols to study cell proliferation by dye dilution, alone or in combination with other methods, has been published [34]. The authors discuss currently available tracking dyes, with suppliers and new spectral properties, as well as describe evaluations to be performed when selecting one for use in multicolor proliferation monitoring. The protocols presented by the authors address the critical issues related Insight into the Leukemia Microenvironment and Cell-cell Interactions Using Flow Cytometry http://dx.doi.org/10.5772/intechopen.76481 27

and more stable. Importantly, what has been already mentioned above, decrease of the fluorescence signal resulting from dye dilution with every division has to be considered when

**Figure 3.** The scheme of tracking cells with proliferation dye. Dilution of CFSE with cell divisions (left panel) and CFSE

In our studies, in which GFP-expressing CML cells have been co-cultured with stroma fibroblasts labeled with CMAC dye, tracking of subpopulations has been combined with analysis of proliferation (**Figure 4**). To do so, subpopulations have been gated based on the fluorescent signal of the cell tracker, either GFP (A) or CMAC (B), followed by separate analysis of cell divisions in each subpopulation. Such strategy allowed to investigate the impact of co-culture conditions on the proliferation index of either leukemia or stromal cells. This protocols can also be utilized in co-cultures under treatment with anticancer drugs to verify the protective effect of stroma. Similar strategy has been used to investigate interactions of lymphocytes with mesenchymal stem cells [31] as well as in the co-cultures of healthy or malign breast tissue-derived stromal cells with human breast adenocarcinoma cell line MCF-7 [32]. Altogether, proliferation dyes represent a very potent group of tracking dyes analyzed with a

Modern flow cytometry, due to development of multiple laser systems, provides possibility for polychromatic analysis of many parameters in single cells. The key to success, particularly in the studies, in which multiple dyes are used to track different types of cell, is therefore to understand the critical issues enabling optimal use of different tracking dyes. It has to be noticed that some limitations appear when using tracking dyes in co-culture experiments. Each class of commercially available cell trackers—lipophilic "membrane" dyes and amino-reactive "protein" dyes possess advantages and limitations. Thus, there is a need for optimization of different proliferation tracking dyes especially when combined together [33]. Recently, a novel edition of protocols to study cell proliferation by dye dilution, alone or in combination with other methods, has been published [34]. The authors discuss currently available tracking dyes, with suppliers and new spectral properties, as well as describe evaluations to be performed when selecting one for use in multicolor proliferation monitoring. The protocols presented by the authors address the critical issues related

tracking cells with proliferation dyes.

detection by flow cytometry (right panel) are shown.

26 Multidimensional Flow Cytometry Techniques for Novel Highly Informative Assays

broad range of available lasers and cytometers.

**3.3. Multiparameter studies**

**Figure 4.** Analysis of cell proliferation estimated upon tracking with CMAC, combined with cell-type gating in co-culture of leukemia and stromal cells. (A) Leukemia cells expressed GFP protein (GFP+) and (B) stromal cells were tracked with CMAC (CMAC+). Labeling with CMAC allowed for cell-type gating and simultaneous analysis of cell divisions. CMAC was detected in the APC channel; histograms show CMAC dilution at different time points of co-culture.

to suboptimal tracking combinations, reproducible labeling, combining of cell tracking dyes with fluorescent antibodies staining and others. Briefly, the recommendations include evaluating the dye's spectral profile and available cytometers/lasers to optimize compatibility with other fluorochromes and to minimize compensation problems, evaluating the effect of labeling on cell growth rate, testing cell divisions and dye dilutions to determine the maximum number of generations to be included when using dye dilution profiles, and verifying that relevant cell functions remain unaltered by tracking dye labeling.

As proliferation is tracked in living cells, it enables to combine analysis of cell proliferation with other live-cell applications, such as immunophenotyping, cell sorting, cell cycle analysis, mitochondrial potential, ROS studies, and others. This possibility has been utilized, for example, in the studies in which myelin-phagocytosing macrophages were co-cultured with CFSE-labeled T lymphocytes followed by analysis of surface receptors and role of nitric oxide [35]. In other studies, cell tracker has been used to investigate immune cells proliferation and cytotoxicity [36].

Another group of trackers is represented by the fixable viability dyes, enabling the post-fixation discrimination of dead cells, which lost plasma membrane integrity before fixation (see **Table 1**, Viability). This gives a novel opportunity to combine them with staining procedures which require fixation. Thus, analysis of internal targets, like as intracellular protein levels or γH2AX, within the population of cells which was alive prior fixation, was possible. In our

CML cells support leukemic cell growth and survival in an autocrine manner, both *in vitro* and *in vivo* [43]. Moreover, factors facilitating leukemic transformation have been identified within vesicles released by leukemia cells [44]. Extracellular vesicles not only directly supported leukemia cell growth but also potently modified components of CML microenvironment to further promote leukemia progression. CML-derived exosomes activated EGFR signaling in stromal cells, which led to IL-8 production by stroma bone marrow fibroblasts and which in turn stimulated leukemia cell adhesion to stromal cells [45, 46]. CML exosomes have been widely studied in terms of their influence on neovascularization process, which has previously been shown to support progression of leukemia [47]. Our own unpublished data suggest that extracellular vesicles released by murine BCR-ABL-expressing progenitor cells may also influence function of cells in distant tissues. Altogether, this supports the notion that leukemic EVs constitute an important mechanism of disease progression both by direct influence on CML cells and by indirect modification of leukemic niche components and immune cells. Moreover, EVs components, such as proteins and microRNAs, have recently become of interest as biomarkers in hematological malignancies [42, 48, 49]. Thus, analysis of EVs number, size, and composition may soon be used to diagnose, stage, and even analyze relapse of hematological malignancies in the clinical

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Multiple studies underline functional relevance of different extracellular vesicles. Thus, a significant aspect of research is analysis of fluorescently labeled EVs uptake or binding to different cells, both in *in vitro* and *in vivo* experiments. It allows to confirm biological relevance of experiments and exclusion of indirect effects on target cells. Different analytical techniques are used to study extracellular vesicles, as there is no one single method which can be proposed to analyze these particles [50]. Confocal microscopy is widely used to analyze EVs uptake by cells [51]. However, the flow cytometry provides a very good and fast method for

Flow cytometry allows to quickly assess if association of EVs with target cells is dose- and timedependent. This may incline biologically active doses of EVs and thus help choose appropriate amounts of EVs for further functional studies. Multicolor flow cytometry allows to monitor association of EVs to different subpopulations of cells, especially *in vivo* or in *ex vivo* cultures of, for example, immune cells. This is doable if a combination of fluorescent EVs labeling is used in parallel with staining of protein markers of subpopulations with fluorochrome-conjugated antibodies. Moreover, flow cytometry analysis enables correlation of EVs uptake with various biological parameters, such as cell viability, apoptosis, proliferation, and expression of signaling molecules. Such approach allows more precise and direct observation of functional effects of EVs. On the other hand, opposed to confocal microscopy, flow cytometry does not distinguish between uptake and binding of extracellular vesicles to target cells, but rather gives information on association of EVs with target cells. Such information may be relevant in terms of mechanism of influence on target cells, as solely binding to surface of target cell suggests regulation through mediators (mostly proteins) on EVs surface rather than by, for example, non-coding RNAs in the lumen.

Various methods and reagents may be used to fluorescently label extracellular vesicles for analysis using flow cytometry (**Table 2**). EVs staining can either lead to labeling of all types of

**4.2. Analysis of EVs uptake and binding by flow cytometry**

quantification of EVs uptake or binding to cells.

setting.

**Figure 5.** Combination of cell tracking by expression of GFP protein (GFP+, green), with analysis of apoptosis by using fixable viability dye and estimation of intracellular level of Grp78 protein (red histograms). Subpopulation expressing high level of GFP was gated (GFP+) and further analyzed. Cells were untreated (control) or treated with antileukemic drug for 24 h.

studies, we have combined cell tracking with GFP expression together with analysis of apoptosis by fixable viability dye and detection of intracellular level of Grp78 protein (**Figure 5**). Cells were co-cultured and the subpopulation expressing high level of GFP (GFP+) was gated and further analyzed. Additionally, as cells were treated with anti-leukemia drug, this procedure enabled to estimate sensitivity to treatment by detection of dead cells.
