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Amine-reactive dyes, also known as LIVE/DEAD® fixable dead cell stains, are a class of viability dyes suitable for identifying dead cells in samples that will be fixed. These dyes cross the cell membranes of dead cells, and react with free amines in the cytoplasm. Live cells exclude these dyes because their cell membranes are intact, and free dye is washed away after staining. Notably, the reaction is irreversible; therefore, when cells are fixed and permeabilized (as with intracellular staining procedures), the bound dye remains associated with the dead cells (unlike other viability dyes). Since amine-reactive dyes are fluorescent when excited by lasers, dead cells can be identified by flow cytometry.
This unit describes procedures, troubleshooting, and outcomes for using the two most commonly used amine-reactive dyes, ViViD and Aqua Blue.
Amine reactive viability dyes offer an innovative alternative to traditional viability markers. There are eight LIVE/DEAD® fixable dead cells stains available in the market today, covering most of the visible and UV spectrum. Two of the commonly used amine reactive dyes, ViViD and Aqua Blue are described in this protocol. These dyes differ from other viability markers because they react with free amines in the cytoplasm. Live cells exclude these dyes because their cell membranes are intact, and free dye is washed away after staining (Figure 1). Notably, the reaction is irreversible; therefore, when cells are fixed and permeabilized (as with intracellular staining procedures), the bound dye remains associated with the dead cells (unlike other viability dyes). To measure these two dyes, it is critical to determine the correct filter configuration for detecting signal from each dye on the flow cytometer. For example, figure 2 shows the excitation curves (dotted lines) and the emission curves (solid lines) of ViViD and Aqua Blue, which can be excited by a 408nm violet laser (vertical black line). The shaded area over the emission curve shows the range of detection. Hence, ViViD is measured between 425-475nm (450/50 band pass filter), while Aqua Blue is detected between 525-575nm (505nm long pass filter + 515/20nm band pass filter). Additionally, it is important to note that the flow cytometer used to measure amine reactive dyes should be calibrated and standardized as described (Perfetto et al., 2006a).
The utility of amine reactive dyes is best demonstrated in the context of staining panels, which employ various combinations of fluorochrome mAb-conjugates, using samples that have been cryopreserved and/or stimulated with antigen. Such samples often include large numbers of dead cells, which can bind m-Ab conjugates non-specifically. In ICS (Intracytoplasmic Cytokine Staining) and peptide-MHC Class I (“tetramer”) assays, (Chattopadhyay et al., 2008; Perfetto et al., 2006b), where detection of rare events is critical, non-specific binding of mAB-conjugated by dead cells may result in significant overestimation of the proportion of antigen-specific cells. In summary, amine reactive viability dyes can be a powerful substitute for more traditional viability dyes and can be used in a variety of panel designs.
The purpose of this protocol is to ensure that an optimal amount of amine reactive dye is used in staining experiments. The optimal concentration is defined as the concentration, which produces the highest signal (MFI) and the lowest background.
To this end, amine reactive dyes should be tested at concentrations above and below the manufacturer's recommended dilution, using samples that contain substantial numbers of dead cells. For example, PBMC samples contain substantial numbers of dead cells when incubated at 37° C for three to five days in RPMI cell culture media (containing 10% serum). Similarly, the frequency of dead cells is high when frozen cells are thawed under sub-optimal conditions (e.g. extended exposure of frozen cells to ambient temperatures). Once titration is completed, new vials from the same lot of amine reactive dye can be used at the same concentration; titration should be performed with each new lot.
The purpose of this protocol is to create a LIVE/DEAD® fixable dye compensation control for correction of spectral overlap in multicolor flow cytometry. The compensation reagents created are reproducible and stable under long-term storage conditions as described in this protocol.
There is one important consideration for using these controls. When dead cells are excluded in the same channel as other cell types (e.g., CD14+ monocytes or CD20+ B-cells, also known as a “DUMP Channel”), only one compensation control is necessary. Typically, the LIVE/DEAD® fixable dye is the brightest reagent in this detector because only the CD3+ T cells are included in the final gating (negative gating), hence only the ViViD stained beads need to be considered as a compensation control. Another advantage of negative gating can be demonstrated in figure 4, where ViViD is combined with anti-CD20 Pacific Blue (PB) and anti-CD14 Pacific Blue to exclude dead cells, B-cells, and monocytes (respectively) using a single detector. After compensation, five populations can be discriminated based on the ViViD and MAB-conjugates (Figure 4A), as follows: live CD3+ T-cells (1), dead CD3+ T-cells (2), monocytes (3), B-cells (4), and live CD3- CD14- CD20- cells (5). Figures 4B-F shows the overlay of the different populations (1-5) over the lymphocyte gate containing the CD3+ T-cells (1). Each population shows the degree of contamination within the traditional lymphocyte gate, dead cells (C), monocytes (D), B-cells (E) and live CD3-14-20- cells (F). Hence, negative cell gating removes many unwanted cells, which might otherwise fall within the gates of interest.
The purpose of this protocol is to demonstrate how to use amine reactive dyes in multicolor cell panels to identify and remove dead cells from the gating analysis. As previously discussed, dead cells can nonspecifically bind mAb-conjugates, which can result in artifacts and erroneous labeling of cell populations (Perfetto et al., 2006b). Hence, only viable cells should be used in most gating analysis strategies. In this protocol, an example of the standard gating strategy using a ViViD dump channel (negative gating) and anti-CD3 will illustrate the utility of the ViViD amine reactive dye to describe gating and analysis strategies.
This section contains all recipes for the protocols, media, beads, etc.
Bead Storage Media:
|Reagent||Stock Conc.||Volume||Final Conc.|
|Fetal calf sera (HI-FCS*)||na||1ml||1%|
|Sodium Azide (NaN3)||5%||400ul||0.02%|
Standard Staining Media:
|Neonatal calf sera (HI NCS*)||20 ml||100%||4%|
|Sodium Azide (NaN3)||0.5ml||20%||0.02%|
|RPMI 1640**||174 ml||n/a||n/a|
Immune monitoring and vaccine immunogenicity studies often require the measurement of low frequency cell populations in samples that have been cryopreserved. This inevitably leads to questions of sensitivity and reproducibility, since dead cells in the sample may non-specifically bind monoclonal antibody-conjugates (mAb-conjugates) and cause significant artifacts (Maecker et al., 2005; O'Brien and Bolton, 1995; Perfetto et al., 2004a; Schmid et al., 1999). Fortunately, viability dyes may be used to exclude dead cells from analysis. Historically, viability dyes were employed that enter damaged cells via compromised cell membranes, and then intercalate into DNA; propidium iodide (PI) is an example of such a dye. However, PI may leak out of cells within a short period of time, leading to significant signal loss (Clarke and Pinder, 1998; Costantino et al., 1995; Desrues et al., 1989). This is particularly problematic when permeablization reagents are used to stain intracellular molecules (such as cytokines), as is often the case in immune monitoring and immunogenecity studies. To avoid this problem, ethidium monoazide (EMA) may be used. This dye covalently binds to DNA after exposure to ultraviolet (UV) light. Although this dye can resolve dead cell populations and is unaffected by intracellular treatments, the need for a UV light source is inconvenient (Riedy et al., 1991). Also, the degree of membrane damage in apoptotic cells can be variable, leaving some Annexin V+ cells with intermediate levels of PI or EMA staining (Matteucci et al., 1999; Waters et al., 2002). The amine reactive dyes as discussed in this protocol avoid many of disadvantages of these traditional viability markers. Hence, these are a good alternative for measuring and removing dead cells from the cell analysis.
The time needed for cell staining with viability dyes and multicolor antibody conjugates is typically no more than 30 minutes. It would be tempting to shorten this procedure by mixing the mAb-conjugates with the viability dye: however, this is not recommended because the staining medium used for antibody staining often contains proteins. Moreover, some antibody preparations typically contain high levels of proteins as stabilizers. These proteins introduce free amines, which compete with free amines from the cytosol of dead cells to bind to the amine reactive dyes. This competition significantly decreases the fluorescence intensity of the dye, and increases background staining. Therefore, we recommend pre-staining cells (resuspended in PBS) with amine reactive dyes, washing the cell sample, and then incubating with multicolor antibodies in the staining medium of choice.
The problem of non-specific binding is particularly notable when analyzing rare events within cryopreserved samples, since these samples are likely to contain significant numbers of dead cells. When viability markers are not included in these analyses, the frequency of the cells of interest can be dramatically misrepresented because dead cells can non-specifically bind reagents. For example, in figure 7, a rare population (CMV-specific CD8+ T-cells) was examined in bone marrow samples. Data was analyzed with and without excluding cells in the dump channel, which included antibodies against CD14 and CD19 (conjugated to Pacific Blue) and ViViD. The top row shows a “traditional” gating strategy (without dump channels) that might be used on a six-parameter flow cytometer tetramer+ cells are identified in a bivariate plot with CD8 after gating to identify T cells on the basis of CD3 expression. The second row shows a polychromatic gating strategy to eliminate aberrant binding events. In this analysis, live CD3+ T cells were distinguished from dead cells, monocytes, and B cells, which could bind tetramer and mAbs nonspecifically. Notably, CD8+ T cells specific for the CMV epitope could be cleanly identified with the polychromatic gating strategy (second row, right panel, 0.62%), whereas tetramer-binding cells are overrepresented in the top panel (1.57%). Backgating analysis shows that many of these cells are binding reagents in the dump channel, suggesting that much of the binding is non-specific (bottom panel; tetramer+ events are shown in blue overlaid on the total population in a bivariate CD3 versus CD14/CD19/ViViD plot). Thus, it is possible that sample-to-sample variation in viability, or in the frequency of B-cells and monocytes, could have a profound impact on the proportion of antigen-specific cells identified, thereby skewing study results. This demonstrates very clearly the need for amine reactive dyes in cell-staining panels.
In some cases, greater intensity of the amine reactive dye can be seen above background but below expected positive intensity. In these situations it is important to determine if these cells should be excluded because they are dead or to include them because they are viable. To this end, the immunophenotype, cytokine production, or proliferation of the ViViD dim and negative cells may be compared. If the vivid dim population shows a similar expression profile as the negative cells, then the former should be included in the analysis. For example, we compared three cell populations sorted from the viability histogram in the first row of figure 8; these include: live CD3+ T cells (ViViD-Low), dim ViViD+ CD3+ cells (ViViD-Mid) and ViViD+ CD3+ dead cells (ViViD-High). In addition to cell surface stains, the cells were loaded with the proliferation marker CFSE. The sorted cells (figure 8, 2nd row) were then cultured in the presence of SEB for five days using standard culture procedures. After this incubation, cells were re-stained for CD3 (anti-CD3-APC) and OrViD (a second amine reactive dye) and measured on the flow cytometer. OrViD positive cells died as a result of the 5-day incubation process and were excluded from the analysis (figure 8, 3rd row). The sorted population of cells that was ViViD-Mid progressed through as many (or more) rounds of division as the ViViD negative population (ViViD-Low), suggesting that these cells should be included in the final analysis (figure 8, 4th row). Notably, ViViD staining is reduced in proliferating cells, presumably as a result of dilution of bound dye into the daughter cells. These results suggest that protein expression is increased for cell populations that are activated in vivo or in vitro resulting in a slightly increased ViViD binding (ViVid Mid) and fluorescence.
The amine reactive dyes have distinct advantages over traditional viability staining. Firstly, they are simple to use, they are stable and they can be used with other mAb-conjugates after the complete interaction with free amines in the cytosol of the dead cell. Secondly, the amine reactive dyes are sold with a variety of emission and excitation wavelengths and can therefore be included in many cells staining panels. This flexibility allows for many traditional mAb-conjugates combinations to remain while “fitting in” a viability dye. These protocols illustrate the utility of the amine reactive dyes in cell biology.
|Dead cells are dimly fluorescent and appear separate poorly from the live cell population.||
|Poor amine reactive dye compensation control. Dim staining as compared to dead cells stained with amine reactive dye.||
|Poor DUMP compensation control. Amine reactive dye appears not to be subtracted||
This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, NIH; the National Cancer Institute, NIH, under contract No. HHSN261200800001E.
*For proprietary reasons, this reagent can only be ordered by phone, and is not available in the Bangs Laboratories catalog.
Disclaimer: The views expressed here are the opinions of the authors and are not to be considered as official or reflecting the views or policies of the Vaccine Research Center / National Institutes of Health / Department of Health and Human Services nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.