BioPath Online

Pathway Focus: Apoptosis Detection

Detection and Quantification of Key Apoptosis Biomarkers
Detecting and Quantifying Key Apoptosis Biomarkers—Multiplex Luminex® Assay
Simplified Apoptosis Detection on Adherent Cells by Flow CytometrySimplified Apoptosis Detection on Adherent Cells by Flow Cytometry—Violet Ratiometric Membrane Asymmetry/Dead Cell Apoptosis Kit

Visualizing DNA FragmentationVisualizing DNA Fragmentation—Click-iT® TUNEL Imaging Assay
Studying Cell DeathStudying Cell Death—Measure Proteins in the Apoptosis Pathway Using ELISAs
High-Purity, High-Bioactivity Proteins for Your ExperimentsHigh-Purity, High-Bioactivity Proteins for Your Experiments—GIBCO® Recombinant Apoptotic Factors

New Antibodies

New Immunoassays

New Molecular Probes® Products

Detecting and Quantifying Key Apoptosis Biomarkers—Multiplex Luminex® Assay

  • Superior performance—Enables accurate, reproducible, and sensitive quantitation of multiple proteins
  • High quality—In-house manufactured antibodies help ensure excellent specificity and sensitivity
  • Fast and easy protocols—Perform and analyze your data in less than one day

To facilitate apoptosis detection, we have developed a multiplex immunoassay for the Luminex® platform that permits the simultaneous detection of several important biomarkers, including cytochrome c, cleaved caspase-3 [175/176], and cleaved poly (ADP-ribose) polymerase (PARP) [214/215]. Multiplex immunoassays allow simultaneous analysis, saving costs,  time, and sample.

The Invitrogen™ Human Apoptosis 3-Plex Assay is a solid-phase sandwich immunoassay designed for simultaneously quantifying the levels of multiple apoptosis proteins within cellular extracts. Beads of defined spectral properties conjugated to analyte-specific capture antibodies and samples (including standards of known analyte concentration, control specimens, and unknowns) are pipetted into the wells of a filter-bottom microplate and incubated for 2 hours. 
During this first incubation, analytes bind to the immobilized (capture) antibodies. After washing, the beads are reacted with a detector antibody mixture for 1 hour. At the end of this incubation, the beads are again washed and then reacted with an R-phycoerythrin (RPE) conjugate solution (containing anti-rabbit RPE and streptavidin RPE) for 30 minutes. During this final incubation, the RPE conjugates bind to the detector antibodies associated with the immune complexes on the beads, forming four-member solid-phase sandwiches. After washing to remove unbound RPE conjugates, the beads are analyzed with the Luminex® 100™ or 200™ instrument. By monitoring the spectral properties of the beads and the amount of associated RPE fluorescence, the concentration of the three analytes can be determined.

  • Learn more about Multiplex Bead-Based Luminex® Technology

Figure 1. Apoptotic pathways.

Figure 2. Nuclear transcription factor protocol.

Figure 3. Apoptosis detection by analyzing various cell fractions. Caspase-3, cytochrome c and PARP were measured in whole Jurkat cell lysates, cytosolic fractions, and mitochondrial fractions, and analyzed using the Invitrogen™ Human Apoptosis 3-Plex Assay. The data presented here demonstrate that treatment for 2 hr with staurosporine resulted in cleavage of caspase-3 and cleavage of the caspase-3 substrate PARP. Increase in cytosolic cytochrome c was accompanied by a decrease in the mitochondrial compartment, consistent with the translocation of this analyte from the mitochondria.

For research use only.  Not intended for human or animal therapeutic or diagnostic use.

Simplified Apoptosis Detection on Adherent Cells by Flow Cytometry—Violet Ratiometric Membrane Asymmetry/Dead Cell Apoptosis Kit

  • Robust assay—Designed for accurate apoptotic analysis on trypsinized cells
  • Easy to use—5-minute incubation, with no wash required
  • Flexible experimental design—Easily combined with other testing reagents

The Violet Ratiometric Membrane Asymmetry Probe⁄Dead Cell Apoptosis Kit provides an easy, efficient method for detecting apoptosis with dead cell discrimination using a violet laser flow cytometer. The Violet Ratiometric Membrane Asymmetry Probe is a novel violet-excitable dye for detecting membrane asymmetry changes during apoptosis. The probe is effective on adherent and suspension cells and correlates with other indicators of apoptosis, such as caspase detection and changes in mitochondrial membrane potential.
The dye exhibits an excited-state intramolecular proton transfer (ESIPT) reaction resulting in dual fluorescence with two emission bands corresponding to 530 and 585 nm, producing a two-color ratiometric response to variations in surface charge. The F2N12S probe is combined with SYTOX® AADvanced Dead Cell Stain, which is capable of passing through the cell membrane only in late-apoptotic or necrotic cells, allowing discrimination from early-apoptotic cells.

Unlike annexin-based assays, the violet ratiometric assay does not require special buffers or wash steps, and is less susceptible to the cell membrane damage commonly found during the physical or chemical removal steps performed when assaying adherent cells, therefore providing better data quality.

Figure 4. Violet Ratiometric Membrane Asymmetry Probe for apoptosis detection. Jurkat cells (T-cell leukemia, human) were treated with 10 μM camptothecin for 4 hr (panels B and D) or left untreated as a control (panels A and C). Samples were analyzed on a flow cytometer with 405 nm excitation using 585 and 530 nm bandpass filters for F2N12S, and 488 nm excitation for SYTOX® AADvanced™ Dead Cell Stain using a 695 nm bandpass filter. Live cells can be discriminated from apoptotic and dead cells by the relative intensities of the two emission bands from F2N12S (A and B). In panels C and D, SYTOX® AADvanced™ Dead Cell Stain fluorescence is plotted against a derived ratio parameter from the two emission bands (585/530 nm) of F2N12S. A = apoptotic cells, L = live cells, D = dead cells.

For research use only.  Not intended for human or animal therapeutic or diagnostic use.

Visualizing DNA Fragmentation—Click-iT® TUNEL Imaging Assay

  • Reliable—Detection of DNA strand breaks using Click-iT® technology
  • Sensitive—Minimal loss of fragile late-apoptotic cells plus low background
  • Multiplex compatible—Allows simultaneous detection of other apoptosis markers

Apoptosis is characterized by a series of biological and structural changes within the cell, ultimately resulting in cell death. Events that occur during apoptosis include permeabilization of the mitochondrial outer membrane, release of cytochrome c from the mitochrondria into the cytosol, activation of caspases, and phosphatidylserine externalization. In addition, structural changes occur as the cell is dismantled and apoptotic bodies form. The Click-iT® TUNEL assay detects fragmentation of genomic DNA, often considered a hallmark of late-stage apoptosis.

The Click-iT® TUNEL assay uses powerful click chemistry—the copper-catalyzed reaction between an alkyne and an azide to form a covalent bond—to detect DNA strand breaks. The modified dUTP molecule (EdUTP) used in this assay contains an alkyne moiety and is readily incorporated at the 3′-OH ends of DNA by the TdT enzyme. Incorporated EdUTP can then be detected by a click reaction with a fluorescently labeled azide. By using small molecule detection, this assay requires only mild fixation and permeabilization conditions.

The Click-iT® TUNEL assay allows researchers to visualize the presence of fragmented DNA at the single-cell level. The assay provides low assay background and the ability to preserve fragile late-apoptotic cells. This reliable and sensitive assay can be easily multiplexed, enabling simultaneous detection of other markers of apoptosis.

Figure 5. Modified nucleotide structures. The alkyne and bromine modifications are significantly smaller than fluorescein.

Figure 6. Detection of apoptosis with the Click-iT® TUNEL Imaging Assay.

Figure 7. DNA strand breaks typical of late-stage apoptosis visualized using the Click-iT® TUNEL Imaging Assay.
HeLa cells were treated with staurosporine, then fixed and permeabilized. The Click-iT® TUNEL Alexa Fluor® 647 Imaging Assay was performed. Activated caspase-3 was detected with a rabbit polyclonal primary antibody for cleaved caspase-3 and labeled with Alexa Fluor® 488 goat anti–rabbit IgG (green fluorescence). Tubulin was detected with a mouse monoclonal anti-tubulin antibody and labeled with Alexa Fluor® 555 goat anti–mouse IgG (orange fluorescence). Nuclei were stained with Hoechst 33342 (blue fluorescence). Coverslips were mounted in ProLong® Gold antifade reagent before imaging. The cells on the right not only have a high level of caspase-3 activity and DNA strand breaks, but also show a loss of structural integrity consistent with cells undergoing apoptosis.

For research use only.  Not intended for human or animal therapeutic or diagnostic use.

Studying Cell Death—Measure Proteins in the Apoptosis Pathway Using ELISAs

  • Sensitive—Measure low levels of apoptosis-related proteins
  • Conserves sample—Small volume of cell lysates needed
  • Fast—Quantitative results in as little as 4 hours

Activated caspase-3, PARP, and cytochrome c are important players in the apoptosis pathway. Apoptosis—a normal process in which cell populations are deleted in response to self-recognition, failure to bind MHC, and cytokine/growth factor withdrawal—regulates and maintains the immune system.

Cytochrome c is a protein that normally resides within the intermitochondrial space that is released to the cytosol in response to apoptotic stimuli. Cleaved caspase-3 [175/176] is an important reporter for initiator caspase activation. Cleaved poly (ADP-ribose) polymerase (PARP) [214/215] is an important reporter for caspase-3 activation. During apoptosis, caspase-3 and caspase-7 cleave PARP between Asp214 and Gly215, which results in a p85 and p25 fragment.
This cleavage process eliminates PARP’s attempt at DNA repair during apoptosis. Therefore, PARP cleavage helps commit cells to the apoptosis pathway and is an ideal marker for examining apoptosis.

ELISAs allow a fast and quantitative method to measure these important apoptotic markers—activated caspase-3, the p85 fragment of PARP, and cytochrome c translocated in the cytosol from the mitochrondrial compartment. The sandwich immunoassay format allows for a very specific measurement using two antibodies against the protein of interest. This format also provides a more sensitive measurement compared to other traditional methods, allowing less cell lysate to be used, and detecting small amounts of protein.

  • Learn more about ELISA Kits & PhosphoELISA® Kits

Figure 8—Apoptosis pathway.

Figure 9. Staurosporine-induced activation of caspase-3 in Jurkat cells. Cells were cultured in RPMI 1640 medium plus 10 % FBS, treated with staurosporine at different concentrations for 5 hr, and lysed with cell extraction buffer. Nontreated cells were used as controls. As shown here, staurosporine induces activation of caspase-3 in a dosage-dependent manner.


Figure 10. Sensitivity comparison of the Cleaved PARP [214/215] ELISA Kit to the Caspase-3 Protease Assay.

For research use only.  Not intended for human or animal therapeutic or diagnostic use.

High-Purity, High-Bioactivity Proteins for Your Experiments—GIBCO® Recombinant Apoptotic Factors

  • Proven compatibility—GIBCO® proteins bioassayed with GIBCO® media
  • High biological activity—More results with less protein
  • High purity—Minimizes interference from other proteins or contaminants
  • Convenience—Proteins can be stocked in your Supply Center

GIBCO® recombinant apoptotic factors are high-purity, high-bioactivity proteins that can be used in combination with GIBCO® specialty media products to stimulate growth of various cell types for use in experimentation. Using GIBCO® apoptosis proteins with their media and cells allows customers to get the accurate and reproducible results they rely on.
The TNF superfamily is perhaps the most widely studied group of apoptotic factors and are important to development and the immune system. Many of these proteins are transmembrane proteins that are subsequently cleaved to form soluble cytokines.

For research use only.  Not intended for human or animal therapeutic or diagnostic use.