Checking Vital Signs: Don’t Let Dead Cells Mislead You

(See a list of the products featured in this article.)

Cell health may not always be the first question on your mind, but the inadvertent inclusion of sick or dead cells in experiments can dramatically affect the outcomes. For instance, including dead cells in immunophenotyping analysis can distort the results, especially for rare phenotypes. Perfetto et al. (2006) showed that light-scatter gating during flow cytometry is not enough to exclude all dead cells from analysis during leukocyte immunophenotyping [1] (Figure 1). Using some of the Invitrogen LIVE/DEAD Fixable Stains listed in the “Cell viability stains” column of Table 1, they were able to efficiently exclude dead cells from analysis and, consequently, significantly increase accuracy in their assays.

We have developed an extensive array of stains and kits to assess cell health and viability. Whether the health of cells is your primary question or simply a critical factor in getting the right answers to other questions, we have a solution for you.

Membrane integrity as a discriminator of live and dead cells

Individual cells are literally defined by their plasma membranes, and loss of membrane integrity is a decisive indicator of cell death. Loss of membrane integrity of cells being analyzed by flow cytometry can be easily detected with either SYTOX Dead Cell Stains or LIVE/DEAD Fixable Dead Cell Stains.

SYTOX Dead Cell Stains are excluded from cells with intact membranes but quickly diffuse into cells that have compromised membranes. Once inside, these dyes bind DNA, which produces a significant enhancement of their fluorescence; live cells remain nonfluorescent and dead cells fluoresce brightly. SYTOX stains are available to match widely available excitation sources (Table 1). These dyes are often used in a “dump channel”, with gating on the viable cells for further analysis (Figure 2). No wash step is required; in fact, SYTOX Dead Cell Stains do not bind covalently to DNA, so dye concentrations must be maintained during analysis.

The LIVE/DEAD Fixable Dead Cell Stains covalently bind available amino acids but are excluded from the cytosol of live, healthy cells. The dyes react with surface proteins of both live and dead cells, but label proteins throughout the cytoplasm of cells with compromised membranes, causing dead cells to fluoresce at least 50 times more brightly than do live cells. Because the labeling is covalent, stained cells can be fixed and permeabilized without losing the viability discrimination signal (Figure 3), making these reagents ideal if you want to fix and permeabilize samples but maintain dead-cell discrimination during subsequent analysis. LIVE/DEAD Fixable Dead Cell Stains are available to match a range of excitation sources and detection channels (Table 1).

Cell cycle analysis

As a cell progresses through the cell cycle, its total DNA content can be used to indicate which stage of the cell cycle it is in. There are two classes of our dyes available to assess cellular DNA content by flow cytometry.

The FxCycle stains, intended for cells that are fixed and permeabilized, provide fluorescence signals proportional to the DNA content of each cell in a population.

The Vybrant DyeCycle dyes offer the ability to stain for DNA content in live cells (Figure 4). VybrantDyeCycledyes are generally used with a viability stain to exclude dead cells from the analysis, but the dyes are not cytotoxic, allowing stained cells to be sorted and then cultured or assessed with functional assays after determining their cell cycle stage.

Measure growing cells by nucleoside incorporation

The growth of cells within a population can be indirectly observed by measuring modified nucleoside incorporation into newly synthesized DNA. EdU (5-ethynyl-2´-deoxyuridine) is a nucleoside analog that is incorporated into DNA during synthesis. The Click-iT EdU Flow Cytometry Assay allows detection of EdU, which contains an alkyne, by a copper-catalyzed reaction that produces a stable covalent bond between the alkyne and a fluorescent dye–labeled azide. The small size of the azide detection reagents means that the fluorescent label has efficient access to the DNA without the need for harsh cell treatment. In the past, DNA synthesis was measured by incorporating the nucleoside analog bromodeoxyuridine (BrdU) into DNA, followed by detection with an anti-BrdU antibody. Although useful in its time, that method requires DNA denaturation (using acid, heat, or DNase) to expose the BrdU to the antibody—a step that can adversely affect sample quality. The Click-iT EdU Flow Cytometry Assay eliminates the need to denature DNA, thus simplifying the assay considerably yet generating comparable results (Figure 5). Click-iT EdU labeling is compatible with fixation protocols.

Monitor cell proliferation via generation analysis

The CellTrace dye family includes carboxyfluorescein diacetate succinimidyl ester (5(6)-CFDA SE, also called CFSE), which spontaneously and irreversibly couples to cellular proteins by reaction with lysine side chains and other available amines. When cells divide, CFSE labeling is distributed equally between the daughter cells, and each successive generation in a population of proliferating cells is marked by a halving of cellular fluorescence intensity (Figure 6). Eight to ten successive generations have been identified this way [2,3]. It is also possible to perform multiplex analysis of CFSE and other probes to correlate cell division status with other cellular function markers [2,4,5]. CellTrace Violet and CellTrace Far Red DDAO-SE dyes provide the same cell generation–marking capability, but have emission maxima of 450 nm and 655 nm, respectively, leaving the green detection channel available for measuring GFP, fluorescein, or other green-fluorescent probes.

Keep track of apoptosis

Apoptosis can be detected by flow cytometry in several ways, and we highlight two approaches here. Caspases are a family of enzymes that play key roles in initiating and effecting apoptosis, and activation of caspase enzymes is a distinctive feature of the early stages of apoptosis. We provides a variety of caspase assays for flow cytometry, including the new CellEvent Caspase-3/7 Green Flow Cytometry Assay Kit, which detects caspase activity with a substrate that, after being cleaved by caspase-3 or caspase-7, binds to DNA and becomes brightly fluorescent (Figure 7).

Another hallmark of apoptosis is the translocation of phosphatidylserine (PS) from the cytoplasmic surface of cell membranes, where it normally resides, to the external surface of cells. We offer a number of flow cytometry–optimized kits that use fluorescently labeled annexin V, a PS-binding protein, to detect PS on the surfaces of cells undergoing apoptosis. Three of those kits are featured in Table 1.

Identify stressed cells

Cells that are environmentally stressed usually contain greatly increased levels of reactive oxygen species (ROS). Invitrogen CellROX reagents are fluorogenic probes developed for the detection and quantitation of ROS in live cells. These cell-permeant reagents are nonfluorescent or very weakly fluorescent in the reduced state; however, when oxidized, they become brightly fluorescent and remain localized within the cell. We offer CellROX Green, CellROX Orange, and CellROX Deep Red Assay Kits validated for flow cytometry. Read more in the article "Oxidative stress from a cell’s point of view".

Table 1. Spectral characteristics of Invitrogen flow cytometry reagents for viability and vitality measurements.

References

1. Perfetto SP, Chattopadhyay PK, Lamoreaux L et al. (2006) J Immunol Methods 313:199–208.
2. Lyons AB, Parish CR (1994) J Immunol Methods 171:131–137.
   
3. Lyons AB (1999) Immunol Cell Biol 77:509–515.
4. Fazekas de St Groth B, Smith AL, Koh WP et al. (1999) Immunol Cell Biol 77:530–538.
5. Hodgkin PD, Lee JH, Lyons AB (1996) J Exp Med 184:277–281.