Checking Vital Signs: Don’t Let Dead Cells Mislead You
Molecular Probes® cell health assays for flow cytometry
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  (Figure 1). Using some of the Molecular Probes® 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.
Life Technologies has developed an extensive array of Molecular Probes® 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.
Figure 1. Exclusion of dead cells eliminates staining artifacts from analysis. After the application of a lymphocyte gate (A), live and dead cells were discriminated using LIVE/DEAD® Fixable Violet Dead Cell Stain Kit (B). Note the significant number of dead cells despite a scatter gate. Subsequent analysis of dead cells (C) and live cells (D) shows the dramatic difference in apparent phenotypes between two cell populations. Reprinted from Perfetto SP, Chattopadhyay PK, Lamoreaux L et al. (2006), with permission from Elsevier .
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).
Figure 2. Viable cell gating with SYTOX® Dead Cell Stains. A mixture of heat-treated and untreated human peripheral blood leucocytes (PBL) was stained with antibody conjugates and 5 nM SYTOX® Red Dead Cell Stain before analysis by flow cytometry using 488 nm and 635 nm excitation. The dot plot showing cells stained for CD3 and CD8 (B) was gated on live cells exhibiting less SYTOX® Red fluorescence (A).
|Figure 3. Use of LIVE/DEAD® Fixable Dead Cell Stains before and after fixation. The LIVE/ DEAD® Fixable Aqua Dead Cell Stain Kit was used to differentially stain a mixture of live (left peak) and heat-treated Jurkat cells (right peak). Cells in (A) were not fixed; cells in (B) were fixed in 3.7% formaldehyde following staining. Samples were analyzed by flow cytometry using 405 nm excitation and ~525 nm emission.|
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 Molecular Probes® 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). Vybrant® DyeCycle™ dyes 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.
|Figure 4. Viable cell gating with Vybrant® DyeCycle™ dyes. Jurkat cells from an overgrown culture were stained with Vybrant® DyeCycle™ Green Stain and then SYTOX® Blue Dead Cell Stain, and analyzed by flow cytometry using 488 nm and 405 nm excitation. The histogram (B) was gated on live cells (A) and shows DNA content distribution in live cells: G0/G1 and G2/M phase peaks are separated by the S phase distribution. Inclusion of the dead cells would have produced aberrant results.|
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.
|Figure 5. Cell proliferation analysis using the Click-iT® EdU Alexa Fluor® 488 Flow Cytometry Assay Kit and FxCycle™ Far Red Stain. (A) Jurkat cells were treated with 10 μM EdU for one hour and stained with Alexa Fluor® 488 azide according to the Click-iT® EdU Alexa Fluor® 488 Flow Cytometry Assay Kit’s protocol, and then analyzed by flow cytometry using 488 nm excitation; clear separation of proliferating cells (which have incorporated EdU) and nonproliferating cells is demonstrated. (B) Jurkat cells were stained with FxCycle™ Far Red Stain and analyzed by flow cytometry using 633 nm excitation. The histogram shows DNA content distribution, with G0/G1 and G2/M phase peaks separated by the S phase distribution. (C) Co-positive staining of cells with the Click-iT® EdU and FxCycle™ stains provides the percentage of cells in S phase (DNA synthesis).|
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.
Figure 6. Cell proliferation analysis. Human peripheral blood lymphocytes were harvested and stained with CellTrace™ CFSE Cell Proliferation Kit on day 0. A portion of the population was arrested at the parent generation using mitomycin C (red peak); the remainder was stimulated with phytohemagglutinin and allowed to proliferate for 5 days. Solid green peaks represent successive generations.
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. Life Technologies 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. Life Technologies offers 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.
|Figure 7. Detection of caspase activity in Jurkat cells using the CellEvent® Caspase-3/7 Green Flow Cytometry Assay Kit. Jurkat cells (human T cell leukemia) were treated with (A) DMSO or (B) 10 µM camptothecin for 3 hr before labeling with the CellEvent® Caspase-3/7 Green Flow Cytometry Assay Kit. Stained samples were analyzed on the Attune® Acoustic Focusing Cytometer equipped with a 488 nm laser, and fluorescence emission was collected using a 530/30 bandpass filter for the CellEvent® reagent and a 690/50 bandpass filter for the SYTOX® AADvanced™ stain (also provided in the kit). Note that treated cells have a higher percentage of apoptotic cells (B) than the basal level of apoptosis seen in the control cells (A). A, apoptotic cells; N, necrotic cells; V, viable cells.|
Identify stressed cells
Cells that are environmentally stressed usually contain greatly increased levels of reactive oxygen species (ROS). Molecular Probes® 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 Molecular Probes® flow cytometry reagents for viability and vitality measurements.
|Emission color||Cell viability stains||Cell cycle dyes|
|SYTOX® Dead Cell Stains||LIVE/DEAD® Fixable Dead Cell Stains||FxCycle™ Stains||Vybrant® DyeCycle™ Stains|
|Excitation: 405 nm laser line|
|Blue||S34857||L34955 (Violet Cell Stain)||F10347 (Violet Stain)||V35003 (Violet Stain)|
|Green||L34957 (Aqua Cell Stain)|
|Orange||L34959 (Yellow Cell Stain)|
|Excitation: 488 nm laser line|
|Red||L23102||F10797 (PI/RNase Stain)|
|Far red||S10349 (AADvanced™ Cell Stain)|
|Excitation: 633 nm or 635 nm laser line|
|Far red||S34859 (Red Cell Stain)||L10120||F10348||V10309 (Ruby Stain)|
|Emission color||Cell proliferation assays||Apoptosis probes||Oxidative stress probes|
|Click-iT® EdU Assays||CellTrace™ Cell Proliferation Kits||CellEvent™ Caspase-3/7 Assay*||Annexin V–Based Apoptosis Assays||CellROX® Kits*|
|Excitation: 405 nm laser line|
|Blue||C10418 (with Pacific Blue™ azide)||C34557 (Violet Stain)||A35136 (with Pacific Blue™ annexin)|
|Excitation: 488 nm laser line|
|Green||C10425 (with Alexa Fluor® 488 azide)||C34554 (with CFSE)||C10427||V13241 (with Alexa Fluor® 488 annexin)||C10492|
|Excitation: 633 nm or 635 nm laser line|
|Far red||C10424 (with Alexa Fluor® 647 azide)||C34553 (with DDAO-SE)||V35113 (with APC annexin)||C10491|
- Perfetto SP, Chattopadhyay PK, Lamoreaux L et al. (2006) J Immunol Methods 313:199–208.
- Lyons AB, Parish CR (1994) J Immunol Methods 171:131–137.
- Lyons AB (1999) Immunol Cell Biol 77:509–515.
- Fazekas de St Groth B, Smith AL, Koh WP et al. (1999) Immunol Cell Biol 77:530–538.
- Hodgkin PD, Lee JH, Lyons AB (1996) J Exp Med 184:277–281.