Telford W, Tamul K, Bradford J (2016) Curr Protoc Cytom 77:9.49.1–9.49.28.
Apoptosis plays a crucial role in the regulation of cell and tissue homeostasis. Well characterized in the immune system, apoptosis mechanisms are now informing anti-cancer therapies. Telford and coauthors recently published a methods chapter providing detailed protocols for several methods to measure and characterize apoptosis using multiparametric flow cytometry. The article provides basic theory discussions, detailed reagent listings, step-by-step procedures, and guidance on analysis methods, while also identifying critical items to consider when performing these assays.
Caspase assays highlighted
A distinctive feature of the early stages of apoptosis is the activation of caspase enzymes, which involves the cleavage of protein substrates. Four assays for measuring caspase activation during cell death are featured: three fluorescence-based assays each label unfixed cells with a fluorogenic substrate (FAM-FLICA, PhiPhiLux™ G1D2, or Invitrogen CellEvent Caspase-3/7 Green Detection Reagent (Figure 1)), and the fourth assay uses an anti-caspase monoclonal antibody, which requires that cells be fixed and permeabilized. Protocols for combining each of the four caspase assays with fluorescent viability stains (e.g., Invitrogen SYTOX Dead Cell Stains and Invitrogen LIVE/DEAD Fixable Dead Cell Stains) and with fluorescent annexin V conjugates in a multiparametric format are provided to more fully characterize cell death.
Figure 1. Detecting active caspases and necrotic 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 Invitrogen CellEvent Caspase 3/7 Green Flow Cytometry Kit. Stained samples were analyzed on the Invitrogen Attune Flow Cytometer equipped with a 488 nm laser, and fluorescence emission was collected using a 530/30 nm bandpass filter for CellEvent Caspase 3/7 Green Detection Reagent and a 690/50 nm bandpass filter for Invitrogen SYTOX AADvanced Dead Cell Stain (both stains are provided in the kit). The 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; V = viable cells; N = necrotic (or late apoptotic) cells.
No one assay fully characterizes apoptosis, and these assays are particularly powerful when combined to determine several characteristics of apoptosis simultaneously. The multiparametric nature of flow cytometry makes the technology ideally suited for measuring apoptosis. As with any flow cytometry experiment, proper controls are essential. A negative (or untreated) control, and a good positive control, are required for proper data interpretation.
Understanding the kinetics of apoptosis is important, as the apoptotic process is highly variable across cell types, and even within the same cell type when cells experience different levels of activation. It is important to measure the cell death process through time, as some cells will die quickly whereas others follow a slower time course of cell death. Care also should be taken when using scatter gating because exclusion of dead and dying cells is not always obvious and submicron-level debris may include information about apoptosis. Keep in mind that apoptosis can be rapid and may continue during assay preparation; therefore, try to keep the assay duration as short as possible. Processing steps like vortex mixing, washing, and centrifugation may induce or accelerate the apoptotic process and may damage or destroy apoptotic cells; therefore, it is recommended that cells be treated gently during sample preparation to avoid these unwanted effects.
Finally, the selected probes must be spectrally compatible and well matched to the cytometer configuration. By choosing fluorescent probes with minimal spectral overlap, color compensation can be minimized. These relatively simple multiparametric assays are powerful techniques for the evaluation and characterization of cell death.
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