Tools to streamline apoptosis research

There are many apoptosis triggers including certain cytokines, protein-protein interactions, and chemicals. Once apoptosis starts, changes in the mitochondria membrane potential can be measured by flow cytometry using the BD™ MitoScreen (JC-1) flow cytometry kit.

Increases in mitochondrial membrane potential lead to increased mitochondrial membrane permeability and the release of soluble proteins such as cytochrome c and pro-caspases.

Caspases are a series of proteases activated upon cleavage at aspartate residues during earliest stages of apoptosis. Active caspases can then cleave many proteins including Poly-ADP ribose polymerase (PARP) and other caspases.

DNA fragmentation is one of the last phases in apoptosis resulting from the activation of endonucleases during the apoptotic process. There are several established methods for the study of DNA fragmentation including isolation and separation of DNA fragments by agarose gel electrophoresis and end labeling.

The BD™ APO-BrdU kit uses end labeling or the terminal deoxynucleotidyl transferase (TdT) nick end labeling (TUNEL method) to support the study of DNA fragmentation. In this assay, TdT catalyzes a template-independent addition of bromolated deoxyuridine triphosphates (Br-dUTP) to the 3'-hydroxyl (OH) termini of double- and single-stranded DNA. After the Br-dUTP is incorporated, these terminal sites of double- and single-stranded DNA are identified using flow cytometry by staining cells with labeled anti-BrdU. In contrast, the BrdU proliferation assay incorporates BrdU into newly synthesized DNA, into sites of DNA strand breaks.

Feature Measured Assays Key Features
Plasma Membrane Alterations
(Phosphatidylserine Exposure)
Annexin binding assay
  • Single conjugates
  • Annexin V kits
  • Detects early apoptosis markers
  • Quick and easy
  • Flow cytometry or immunofluorescence application
Mitochondrial Changes BD MitoScreen Kit Fast, easy, single cell resolution by flow cytometry or fluorescent microscopy
Caspase Activation Caspase Activity Assay Kits and Reagents Quick and easy, uses spectrofluorometry
  Active Caspase-3 immunoassays ELISA, flow cytometry, or Western blot
DNA Fragmentation
  • APO-BrdU TUNEL Assay
Works with adherent cells, single cell resolution in conjunction with cell cycle analysis by flow cytometry

With an overwhelming number of available techniques and products, selecting the most appropriate method is often difficult. To help make this choice easier, the overview above summarizes commercially available assays from a biological perspective.

Measurement of Cleaved Caspases and PARP

Caspases are important initiators of apoptosis. One of the earliest and most consistently observed characteristics of apoptosis is the activation of a series of cytosolic proteases, called caspases. When apoptosis is activated, caspases cleave multiple protein substrates en masse, which leads to the loss of cellular structure and function, and ultimately results in cell death. In particular, caspases -8, -9, and -3 have been implicated in apoptosis: caspase-9 in the mitochondrial pathway, caspase-8 in the Fas/CD95 pathway, and caspase-3 more downstream, activated by multiple pathways.

Additional Techniques for the Detection of Apoptosis

BD Biosciences carries a variety of reagents to measure caspases, particularly caspase-3. They include antibodies directed exclusively against the active form of the caspase. These antibodies are available in a variety of formats and can be used for flow cytometry, imaging, ELISA, and Western blot.

BD Biosciences offers a range of tools for caspase activity assays from individual fluorogenic peptide substrates and inhibitors, to kits, to ready-to-use assay plates. All are based on the use of synthetic tetrapeptide substrates that are designed such that proteolytic cleavage by active human or mouse caspases results in release of a fluorophore or chromophore. The individual synthetic tetrapeptide substrates, together with the caspase inhibitors and active caspase enzymes, offer flexibility in the experimental design of a caspase activity assay.

Caspase-3 cleavage/inhibition reactions
Caspase-3 cleavage/inhibition reactions

Active caspase-3 binds to the fluorogenic Ac-DEVD-AMC substrate and cleaves it between asparatic acid (D) and AMC, releasing the fluorescent AMC. AMC fluorescence is quantified by UV spectrofluorometry. The Ac-DEVD-CHO aldehyde inhibitor binds strongly to the caspase-3 active site and blocks substrate binding. Hence, Ac-DEVD-AMC is not cleaved and fluorescence is not emitted.

Flow cytometric analysis of apoptotic and non-apoptotic populations using anti-active caspase-3 antibodies.
Flow cytometric analysis of apoptotic and non-apoptotic populations using anti-active caspase-3 antibodies.

Jurkat T cells (A, A1) or mouse thymocytes (B, B1) were left untreated (A, B) or treated for 4 h with camptothecin (A1) or a mouse Fas monoclonal antibody, clone Jo2 (Cat. No. 554254) to induce apoptosis (B1). Cells were permeabilized and then stained with PE-conjugated active caspase-3 antibodies (Cat. No. 557091). Untreated cells were primarily negative for the presence of active caspase-3, whereas about half of each population of cells induced to undergo apoptosis had detectable active caspase-3.