Eliminate Dead Cells for Increased Accuracy
Because dead cells tend to bind nonspecifically to many reagents, they often give rise to false positive results in flow cytometry. Thus, identifying and removing data points representing dead cells is a critical step in ensuring accurate results. Cell viability assays help researchers to distinguish live and dead cell populations more accurately than by relying on scatter characteristics alone. This article reviews two types of Molecular Probes® dead cell assays for flow cytometry: the SYTOX® series of impermeant DNA dyes and the LIVE/DEAD® fixable dead cell stains.
Nucleic Acid Dead Cell Stains
Loss of membrane integrity is a definitive indicator of cell death in flow cytometric assays. Cells that exclude a dead cell dye are considered viable, while cells with a compromised membrane allow the dye inside into cell to stain an internal component, thus identifying the cell as dead. The SYTOX® dead cell stains are impermeant nucleic acid dyes that enter a dead cell and bind to DNA, where they undergo a significant fluorescence enhancement (Figures 1 and 2) [1–6]. SYTOX® dyes provide bright, accurate dead cell discrimination in a variety of color options to accommodate any experimental design.
Because the SYTOX® dyes bind in equilibrium with DNA, external dye concentration must be maintained during analysis. SYTOX® dyes should be added last in any staining protocol and should not be washed out before data acquisition. Impermeant DNA dyes are not compatible with fixation or intracellular staining protocols.
Figure 1. Dead cells can nonspecifically bind to antibodies, giving rise to inaccurate results. Lysed human peripheral blood from a normal donor was labeled with CD4–Pacific Blue™ and CD8–Pacific Orange™ direct conjugates, and then labeled with the SYTOX® Orange Dead Cell Stain Kit before acquisition on the Attune® Acoustic Focusing Cytometer. For SYTOX® Orange dye, 488 nm excitation was used with a 575/24 bandpass filter; for Pacific Blue™ dye, 405 nm excitation was used with a 450/40 bandpass; for Pacific Orange™ dye, 405 nm excitation was used with a 603/48 bandpass. (left) The plot was gated on lymphocytes with all cells (live and dead) included; an abnormal co-positive population is observed when including dead cells in the analysis. (right) The plot was first gated on SYTOX® Orange–negative cells (live cells only) and then on a live lymphocyte gate; the mutually exclusive CD4 and CD8 populations are accurately identified by gating on live cells.
|Figure 2. Dead cell discrimination using SYTOX® stains. Jurkat cells (human T cell leukemia) were treated with 10 μM camptothecin for 4 hr, then labeled with the Violet Annexin V/Dead Cell Apoptosis Kit using Pacific Blue™ annexin V and SYTOX® AADvanced™ dead cell stain, and analyzed by flow cytometry using 488 nm excitation on the Attune® Acoustic Focusing Cytometer with 530/30 and 575/24 bandpass filters. Live cells (green), apoptotic cells (blue), and necrotic cells (red) are distinguishable.|
Fixable Dead Cell Stains
Like the SYTOX® dyes, the LIVE/DEAD® Fixable Dead Cell Stain Kits utilize the loss of membrane integrity for dead cell discrimination (Figure 3). However, the LIVE/DEAD® fixable kits are designed to assess cell viability in samples after fixation and/or permeabilization (Figure 4). Often referred to as amine-reactive dyes, these stains are based on the reaction of a fluorescent reactive dye with cellular proteins [7–10]. In live cells, only surface proteins bind to the reactive dye, resulting in dim fluorescence. The reactive dye can enter dead cells and label proteins in the interior of the cell, producing at least a 50-fold increase in fluorescence.
Because the LIVE/DEAD® dyes react covalently with proteins, dead cell discrimination is completely preserved following fixation of the sample with formaldehyde under conditions that are commonly used for intracellular immunophenotyping and to inactivate pathogens. There are eight colors of amine-reactive dyes to choose from, offering flexibility in experimental design.
Figure 3. Exclusion of dead cells eliminates staining artifacts from analysis. After the application of a lymphocyte gate (A), live and dead cells were discriminated using the LIVE/DEAD® Fixable Violet Dead Cell Stain Kit (B). Subsequent analysis of dead (C) and live (D) cells shows the dramatic difference in apparent phenotypes between two cell populations. Reprinted from J Immunol Methods (2006) 313:199, with permission from Elsevier.
Figure 4. Dead cells can be identified after fixation and permeabilization. Jurkat cells were treated with 10 µM EdU for 2 hr, labeled with the LIVE/DEAD® Fixable Violet Dead Cell Stain Kit, then fixed and permeabilized before labeling with Click-iT® EdU Alexa Fluor® 488 azide and FxCycle™ Far Red stain. (A) Live (green) and dead (red) cells as identified by the LIVE/DEAD® amine-reactive dye. (B) The plot was gated on live and dead cells combined. (C) The plot was gated only on live cells. The accuracy of results is improved by eliminating dead cells from the analysis.
Improve the Accuracy of Your Flow Cytometry Data
Removing data points representing dead cells is a critical step for accurate flow cytometry analysis. Molecular Probes® dead cell assays allow accurate discrimination in a variety of bright fluorescence options for flexible experimental design (Table 1).
Table 1. Cell Viability Assay Selection Guide.
|Laser||Traditional DNA stains||Fixable stains|
|UV||• SYTOX® Blue (450/50*)||• LIVE/DEAD® fixable blue dead cell stain (350/40*)|
|405 nm||• SYTOX® Blue (450/50*)||• LIVE/DEAD® fixable violet dead cell stain (450/40*)
• LIVE/DEAD® fixable aqua dead cell stain (530/50*)
• LIVE/DEAD® fixable yellow dead cell stain (585/42*)
|488 nm||• SYTOX® Green (530/30*)
• SYTOX® AADvanced™ (>650*)
|• LIVE/DEAD® fixable green dead cell stain (530/30*)
• LIVE/DEAD® fixable red dead cell stain
(>650 or 600/20*)
|532 nm||• SYTOX® Orange (585/42*)
• SYTOX® AADvanced™ (>650*)
|• LIVE/DEAD® fixable red dead cell stain
(>650 or red bandpass*)
|561 nm||• LDS 751 (700/20*)||• LIVE/DEAD® fixable red dead cell stain
(>650 or red*)
|633/5 nm||• SYTOX® Red (660/20*)||• LIVE/DEAD® fixable far-red dead cell stain (660/20*)
• LIVE/DEAD® fixable near-IR dead cell
|*Recommended filters (nm)|
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