Assays for Apoptosis and Autophagy—Section 15.5

Page Contents

• Apoptosis Assays Using Nucleic Acid Stains
• Apoptosis Assay that Detect DNA Strand Breaks
• Apoptosis Assays that Detect Membrane Asymmetry
• Apoptosis Assays Based on Protease Activity
• Apoptosis Assays Using Mitochondrial Stains
• Assays for Autophagy
• Data Table
• Ordering Information

Apoptosis (programmed cell death) is the genetically controlled ablation of cells during normal development. Inappropriately regulated apoptosis is implicated in disease states such as Alzheimer disease, stroke and cancer. Apoptosis is distinct from necrosis in both the biochemical and the morphological changes that occur. In contrast to necrotic cells, apoptotic cells are characterized morphologically by compaction of the nuclear chromatin, shrinkage of the cytoplasm and production of membrane-bound apoptotic bodies. Biochemically, apoptosis is distinguished by fragmentation of the genome and cleavage or degradation of several cellular proteins.

As with cell viability, no single parameter fully defines cell death in all systems; therefore, it is often advantageous to use several different approaches when studying apoptosis. Several methods have been developed to distinguish live cells from early and late apoptotic cells and from necrotic cells; these are described below and in a number of review articles. Anti-cancer drug candidates failing to induce apoptosis are likely to have decreased clinical efficacy, making apoptosis assays important tools for high-throughput drug screening.

DNA Stains for Detecting Apoptotic Cells

The characteristic breakdown of the nucleus during apoptosis comprises collapse and fragmentation of the chromatin, degradation of the nuclear envelope and nuclear blebbing, resulting in the formation of micronuclei. Therefore, nucleic acid stains can be useful tools for identifying even low numbers of apoptotic cells in cell populations. Several nucleic acid stains, all of which are listed in Nucleic Acid Stains—Section 8.1, have been used to detect apoptotic cells by fluorescence imaging or flow cytometry.

DNA fragmentation can also be detected in vitro using electrophoresis. DNA extracted from apoptotic cells and then separated by gel electrophoresis reveals a characteristic ladder pattern of low molecular weight DNA fragments. Our ultrasensitive SYBR Green I nucleic acid stain (S7567, Nucleic Acid Detection on Gels, Blots and Arrays—Section 8.4) allows the detection of even fewer apoptotic cells in these applications ().

Membrane Permeability/Dead Cell Apoptosis Kit

Our Membrane Permeability/Dead Cell Apoptosis Kit (V13243) detects apoptosis based on changes that occur in the permeability of cell membranes. This kit contains ready-to-use solutions of both the YO-PRO-1 and propidium iodide nucleic acid stains. Our YO-PRO-1 nucleic acid stain (also available as a stand-alone reagent, Y3603) selectively passes through the plasma membranes of apoptotic cells and labels them with moderate green fluorescence. The dyes included in this kit are effectively excited by the 488 nm spectral line of the argon-ion laser and are useful for both flow cytometry (Figure 15.5.1) and fluorescence microscopy (). The kit components, number of assays and assay principles are summarized in Molecular Probes apoptosis assay kits—Table 15.4.

Figure 15.5.1 Flow cytometric analysis of Jurkat cells using the Membrane Permeability/Dead Cell Apoptosis Kit (V13243). Jurkat human T-cell leukemia cells were first exposed to10 µM camptothecin for four hours (top panel) or left untreated (as control, bottom panel). Cells were then treated with the reagents in the kit and analyzed by flow cytometry. Note that the camptothecin-treated cells (top panel) have a significantly higher percentage of apoptotic cells (indicated by an "A") than the basal level of apoptosis seen in the control cells (bottom panel). V = viable cells, D = dead cells.

Chromatin Condensation/Dead Cell Apoptosis Kit

Chromatin Condensation/Dead Cell Apoptosis Kit (V13244) provides a rapid and convenient assay for apoptosis based upon fluorescence detection of the compacted state of the chromatin in apoptotic cells. This kit contains ready-to-use solutions of the blue-fluorescent Hoechst 33342 dye (excitation/emission maxima ~350/461 nm when bound to DNA), which stains the condensed chromatin of apoptotic cells more brightly than the chromatin of nonapoptotic cells, and the red-fluorescent propidium iodide (excitation/emission maxima ~535/617 nm when bound to DNA), which is permeant only to dead cells with compromised membranes. The staining pattern resulting from the simultaneous use of these dyes makes it possible to distinguish normal, apoptotic and dead cell populations by flow cytometry or fluorescence microscopy. The 351 nm spectral line of an argon-ion laser or other suitable UV source is required for excitation of the Hoechst 33342 dye, whereas propidium iodide can be excited with the 488 nm spectral line of an argon-ion laser. The kit components, number of assays and assay principles are summarized in Molecular Probes apoptosis assay kits—Table 15.4.

Chromatin Condensation/Membrane Permeability/Dead Cell Apoptosis Kit

Chromatin Condensation/Membrane Permeability/Dead Cell Apoptosis Kit (V23201) combines the detection principles used in the two related kits described above. Three nucleic acid stains—Hoechst 33342, YO-PRO-1 and propidium iodide—are utilized to identify the blue-fluorescent live-cell population, the green-fluorescent apoptotic population and the red-fluorescent dead-cell population by flow cytometry. The stains are provided as separate solutions to facilitate optimization of the assay for the cell line under study and the equipment available. Once optimized, however, the assay can be completed using simultaneous staining with a mixture of the three nucleic acid stains and either UV excitation of all three dyes or with a combination of UV excitation for the Hoechst 33342 dye and excitation by the 488 nm spectral line of the argon-ion laser. The kit components, number of assays and assay principles are summarized in Molecular Probes apoptosis assay kits—Table 15.4.

Violet Membrane Permeability/Dead Cell Apoptosis Kit

Like the Membrane Permeability/Dead Cell Apoptosis Kit, our Violet Membrane Permeability/Dead Cell Apoptosis Kit (V35123) detects apoptosis based on changes that occur in the permeability of cell membranes (Molecular Probes apoptosis assay kits—Table 15.4). This kit contains ready-to-use solutions of both PO-PRO-1 and 7-aminoactinomycin (7-AAD) nucleic acid stains. Our PO-PRO-1 nucleic acid stain (also available as a stand-alone reagent, P3581) selectively passes through the plasma membranes of apoptotic cells and labels them with violet fluorescence. Furthermore, annexin V labeling of apoptosis yields poor results with trypsinized cells, whereas PO-PRO-1 dye provides the same efficiency for detecting apoptosis with trypsinized cells as it does with suspension cells. Necrotic cells are stained with the red-fluorescent 7-AAD, a DNA-selective dye that is membrane impermeant but that easily passes through the compromised plasma membranes of necrotic cells. Live cells are not appreciably stained by either PO-PRO-1 or 7-AAD. The dyes included in this kit are effectively excited by a flow cytometer that uses both the 405 nm spectral line of the violet laser and the 488 nm spectral line of the argon-ion laser for excitation. The kit components, number of assays and assay principles are summarized in Molecular Probes apoptosis assay kits—Table 15.4.

Comet (Single-Cell Gel Electrophoresis) Assay to Detect Damaged DNA

The Comet assay, or single-cell gel electrophoresis assay, is used for rapid detection and quantitation of DNA damage from single cells. The Comet assay is based on the alkaline lysis of labile DNA at sites of damage. Cells are immobilized in a thin agarose matrix on slides and gently lysed. When subjected to electrophoresis, the unwound, relaxed DNA migrates out of the cells. After staining with a nucleic acid stain, cells that have accumulated DNA damage appear as fluorescent comets, with tails of DNA fragmentation or unwinding (). In contrast, cells with normal, undamaged DNA appear as round dots, because their intact DNA does not migrate out of the cell. The ease and sensitivity of the Comet assay has provided a fast and convenient way to measure damage to human sperm DNA, evaluate DNA replicative integrity, monitor the sensitivity of tumor cells to radiation damage and assess the sensitivity of molluscan cells to toxins in the environment. The Comet assay can also be used in combination with FISH to identify specific sequences with damaged DNA. Comet assays have traditionally been performed using ethidium bromide to stain the DNA; however, use of the SYBR Gold and SYBR Green I stains improves the sensitivity of this assay ().

Click-iT TUNEL Alexa Fluor Imaging Assays

The terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL) assay—based on the incorporation of modified dUTPs by terminal deoxynucleotidyl transferase (TdT) at the 3'-OH ends of fragmented DNA—is probably the most widely used in situ test for studying apoptotic DNA fragmentation. For a sensitive and reliable TUNEL imaging assay, it is vital that the modified nucleotide is an efficient substrate for TdT. We have developed a Click-iT TUNEL imaging assay that incorporates an alkyne-modified dUTP (Figure 15.5.2) at the 3'-OH ends of fragmented DNA using TdT and then detects the enzymatically incorporated nucleotide using a copper (I)–catalyzed click reaction with an azide-derivatized fluorophore (Figure 15.5.3).

The Click-iT labeling reaction is based on a copper-catalyzed azide–alkyne cyloaddition and derives its high degree of specificity from the fact that the azide and alkyne reaction partners have no endogenous representation in biological molecules, cells, tissues or model organisms. The minimally modified EdUTP nucleotide (Figure 15.5.2) used in the Click-iT TUNEL imaging assay is rapidly incorporated by TdT, allowing samples to be rapidly fixed in order to preserve late-stage apoptotic cells, thereby lessening the possibility of false-negative results due to cell detachment and subsequent loss. Compared with assays that use one-step incorporation of dye-modified nucleotides, the fast and reliable Click-iT TUNEL imaging assay can detect a higher percentage of apoptotic cells under identical conditions in two hours or less (Figure 15.5.4). The Click-iT TUNEL Imaging Assay Kits are available with a choice of azide-derivatized Alexa Fluor dyes, providing flexibility for combination with other apoptosis detection reagents. They include:

• Click-iT TUNEL Alexa Fluor 488 Imaging Assay (C10245)
• Click-iT TUNEL Alexa Fluor 488 Imaging Assay (C10246)
• Click-iT TUNEL Alexa Fluor 488 Imaging Assay (C10247)

The Click-iT TUNEL assays have been tested in HeLa, A549 and CHO K1 cells with a variety of reagents that induce apoptosis, including staurosporine, and multiplexed with antibody-based detection of other apoptosis biomarkers such as cleaved poly(ADP-ribose) polymerase (PARP), cleaved caspase-3 and phosphohistone 2B. It has also proven effective for detection of apoptosis induced by siRNA knockdown of the DEC2 transcription factor in human MCF-7 breast cancer cells. Click-iT labeling technology and the details of the click reaction are discussed in Click Chemistry—Section 3.1. For a complete list azide and alkyne derivatives compatible with Click-iT labeling technology, see Molecular Probes azide and alkyne derivatives—Table 3.1.

Figure 15.5.2 The EdUTP nucleotide, provided in the Click-iT TUNEL Imaging Assay Kits.

Figure 15.5.3 Detection of apoptosis with the Click-iT TUNEL imaging assay.

Figure 15.5.4 TUNEL assay comparison—percentage positives detected. HeLa cells were treated with 0.5 μM staurosporine for 4 hours. Following fixation and permeabilization, TUNEL imaging assays were performed according to the manufacturer's instructions. The percent positives were calculated based upon the corresponding negative control. Imaging and analysis was performed using a Thermo Fisher Scientific Cellomics ArrayScan II.

APO-BrdU TUNEL Assay Kit

Because DNA fragmentation is one of the most reliable methods for detecting apoptosis, we have collaborated with Phoenix Flow Systems to offer the APO-BrdU TUNEL Assay Kit (A23210), which provides all the materials necessary to label and detect the DNA strand breaks of apoptotic cells. When DNA strands are cleaved or nicked by nucleases, a large number of 3'-hydroxyl ends are exposed. In the APO-BrdU assay, these ends are labeled with BrdUTP and terminal deoxynucleotidyl transferase (TdT) using the TUNEL technique described above. Once incorporated into the DNA, BrdU is detected using an Alexa Fluor 488 dye–labeled anti-BrdU monoclonal antibody (). This kit also provides propidium iodide for determining total cellular DNA content, as well as fixed control cells for assessing assay performance.

The APO-BrdU TUNEL Assay Kit includes complete protocols for use in flow cytometry applications, though it may also be adapted for use with fluorescence microscopy. Each kit contains:

• Terminal deoxynucleotidyl transferase (TdT), for catalyzing the addition of BrdUTP at the break sites
• 5-Bromo-2'-deoxyuridine 5'-triphosphate (BrdUTP)
• Alexa Fluor 488 dye–labeled anti-BrdU mouse monoclonal antibody PRB-1, for detecting BrdU labels
• Propidium iodide/RNase staining buffer, for quantitating total cellular DNA
• Reaction, wash and rinse buffers
• Positive control cells (a fixed human lymphoma cell line)
• Negative control cells (a fixed human lymphoma cell line)
• Detailed protocols (APO-BrdU TUNEL Assay Kit) 

Sufficient reagents are provided for approximately 60 assays of 1 mL samples, each containing 1–2 × 10⁶ cells/mL.

Detecting DNA Strand Breaks with ChromaTide Nucleotides

Break sites have traditionally been labeled with biotinylated dUTP, followed by subsequent detection with an avidin or streptavidin conjugate (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6, Molecular Probes avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates—Table 7.9). However, a more direct approach for detecting DNA strand breaks in apoptotic cells is possible via the use of our ChromaTide BODIPY FL-14-dUTP (C7614) as a TdT substrate (). The single-step BODIPY FL dye–based assay has several advantages over indirect detection of biotinylated or haptenylated nucleotides, including fewer protocol steps and increased cell yields. BODIPY FL dye–labeled nucleotides have also proven superior to fluorescein-labeled nucleotides for detection of DNA strand breaks in apoptotic cells because they provide stronger signals, a narrower emission spectrum and less photobleaching ().

In situ DNA modifications by labeled nucleotides have been used to detect DNA fragmentation in what may be apoptotic cells in autopsy brains of Huntington's and Alzheimer disease patients. DNA fragmentation is also associated with amyotrophic lateral sclerosis. Analogous to TdT's ability to label double-strand breaks, the E. coli repair enzyme DNA polymerase I can be used to detect single-strand nicks, which appear as a relatively early step in some apoptotic processes. Because our ChromaTide BODIPY FL-14-dUTP (C7614) and ChromaTide fluorescein-12-dUTP (C7604) are incorporated into DNA by E. coli DNA polymerase I, they are also effective for in situ labeling with the nick translation method.

High-Content Analysis of Genotoxicity and Cytotoxicity

In mammalian cells, a double-strand break (DSB) in genomic DNA is a potentially lethal lesion. One of the earliest known responses to DSB formation is phosphorylation of H2A histones. Specifically, DNA damaging agents induce phosphorylation of histone variant H2AX at Ser139, leading to the formation of DNA foci at the site of DNA DSBs.

The HCS DNA Damage Kit (H10292) was developed to enable simultaneous quantitation of two cell health parameters, genotoxicity and cytotoxicity, by high-content analysis in the same cell (Figure 15.5.5). DNA damage is measured as an indication of genotoxicity and accomplished by specific antibody-based detection of phosphorylated H2AX (Ser139) in the nucleus. Cytotoxicity is measured with the Image-iT DEAD Green viability stain (also available as a stand-alone reagent, I10291), a cell-impermeant, nonfluorescent, high-affinity DNA stain that forms highly fluorescent and stable dye-nucleic acid complexes when bound to DNA. Thus, staining of nuclear DNA by the Image-iT DEAD Green viability stain cannot occur in live cells due to the impermeability of the plasma membrane to the stain. Drugs and test compounds that lead to serious cell injuries, including plasma membrane permeability, allow entry of the Image-iT DEAD Green viability stain, enabling discrimination of dead cells. Hoechst 33342, which stains nuclear DNA in live and dead cells, is included in the kit as a segmentation tool for automated image analysis.

The HCS DNA Damage Kit contains sufficient material to perform the DNA damage assay on two 96-well plates when used as described in the protocol provided.

• Image-iT Dead Green viability stain
• pH2AX mouse monoclonal antibody
• Alexa Fluor 555 goat anti–mouse IgG antibody
• Hoechst 33342 nucleic acid stain
• Detailed protocols (HCS DNA Damage Kit)

Figure 15.5.5 Detection of genotoxicity and cytotoxicity in valinomycin-treated A549 cells using the HCS DNA Damage Kit (H10292). A549 cells were treated with 30 µM or 120 µM valinomycin for 24 hr before performing the assay. With increasing concentrations of valinomycin, cells showed genotoxic effects as indicated by detection with a pH2AX antibody in conjunction with Alexa Fluor 555 goat anti–mouse IgG antibody (orange fluorescence), and cytotoxic effects as indicated by staining with the Image-iT DEAD Green viability stain (green fluorescence). Blue-fluorescent Hoechst 33342 was used as a nuclear segmentation tool, and Alexa Fluor 647 phalloidin was used to visualize F-actin (pseudocolored magenta). The image on the left shows untreated cells with intact F-actin cytoskeletons and no evidence of cytotoxicity or genotoxicity. The image on the right shows cells treated with 120 µM valinomycin, which completely disrupted the actin cytoskeletons, increased levels of DNA damage and compromised plasma membrane integrity.

Violet Ratiometric Membrane Asymmetry Probe/Dead Cell Apoptosis Kit

The Violet Ratiometric Membrane Asymmetry Probe/Dead Cell Apoptosis Kit (A35137) provides a simple and fast method for detecting apoptosis with dead-cell discrimination by flow cytometry. The violet ratiometric membrane asymmetry probe F2N12S (4'-N,N-diethylamino-6-(N-dodecyl-N-methyl-N-(3-sulfopropyl))ammoniomethyl-3-hydroxyflavone, ) is a novel violet diode–excitable dye for the detection of membrane phospholipid asymmetry changes during apoptosis. This dye exhibits an excited-state intramolecular proton transfer (ESIPT) reaction, resulting in a dual fluorescence with two emission bands corresponding to 530 nm and 585 nm and producing a two-color ratiometric response to variations in surface charge. This ratiometric probe is therefore a self-calibrating indicator of apoptotic transformation, which is independent of probe concentration, cell size and instrument variation, such as fluctuations of laser intensity or sensitivity of the detectors.

Given that apoptosis modifies the surface charge of the outer leaflet of the plasma membrane, F2N12S can be used to monitor changes in membrane asymmetry that occur during apoptosis through a change in the relative intensity of the two emission bands of the dye (Figure 15.5.6). The F2N12S-based apoptosis assay allows samples to be analyzed after a 5-minute incubation at room temperature and does not require special buffers or wash steps. This kit can be paired with other reagents such as MitoProbe DiIC₁(5) or annexin V for multiparametric analysis of apoptosis and viability. The kit components, number of assays and assay principles are summarized in Molecular Probes apoptosis assay kits—Table 15.4.

Figure 15.5.6 Jurkat cells (T-cell leukemia, human) treated with 10 μM camptothecin for four hours (panels B and D) or untreated control (panels A and C). Cells were stained according to the protocol. Samples were analyzed on a flow cytometer with 405 nm excitation using 585 nm and 530 nm bandpass filters for F2N12S and 488 nm excitation for SYTOX AADvanced™ dead cell stain using a 695 nm bandpass filter. In panels A and B, living cells can be discriminated from apoptotic and dead cells by the relative intensities of the two emission bands from F2N12S. 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.

Annexin V Conjugates

The human vascular anticoagulant annexin V is a 35–36 kilodalton, Ca²⁺-dependent phospholipid-binding protein that has a high affinity for the anionic phospholipid phosphatidylserine (PS). In normal viable cells, phosphatidylserine is located on the cytoplasmic surface of the cell membrane. In apoptotic cells, however, phosphatidylserine is translocated from the inner to the outer leaflet of the plasma membrane, exposing phosphatidylserine to the external cellular environment where it can be detected by annexin V conjugates. In leukocyte apoptosis, phosphatidylserine on the outer surface of the cell marks the cell for recognition and phagocytosis by macrophages.

Highly fluorescent annexin V conjugates provide quick and reliable detection methods for studying the externalization of phosphatidylserine, an indicator of intermediate stages of apoptosis. Nuclear fragmentation, mitochondrial membrane potential flux and caspase-3 activation apparently precede phosphatidylserine "flipping" during apoptosis, whereas permeability to propidium iodide and cytoskeletal collapse occur later. The difference in fluorescence intensity between apoptotic and nonapoptotic cells stained by our fluorescent annexin V conjugates, as measured by flow cytometry, is typically about 100-fold (Figure 15.5.8). Our annexin V conjugates are available as stand-alone reagents, each suitable for 50–100 flow cytometry assays or many more imaging assays, or in several variations of our apoptosis assay kits (Molecular Probes apoptosis assay kits—Table 15.4). We also offer a concentrated annexin-binding buffer (V13246) that facilitates the binding of annexin V to phosphatidylserine in apoptosis assays. Our annexin V conjugates include:

• Alexa Fluor 488 annexin V (A13201, )
• Fluorescein (FITC) annexin V (A13199)
• Oregon Green 488 annexin V (A13200)
• Alexa Fluor 555 annexin V (A35108)
• R-phycoerythrin (R-PE) annexin V (A35111)
• Alexa Fluor 568 annexin V (A13202)
• Alexa Fluor 594 annexin V (A13203)
• Alexa Fluor 647 annexin V (A23204)
• Allophycocyanin (APC) annexin V (A35110)
• Alexa Fluor 680 annexin V (A35109)
• Alexa Fluor 350 annexin V (A23202)
• Pacific Blue annexin V (A35122)
• Biotin-X annexin V (A13204)

Single Channel Annexin V/Dead Cell Apoptosis Kit

With the Single Channel Annexin V/Dead Cell Apoptosis Kit (V13240), apoptotic cells are detected based on the externalization of phosphatidylserine. This kit contains recombinant annexin V conjugated to the Alexa Fluor 488 dye, our brightest and most photostable green fluorophore, to provide maximum sensitivity. In addition, the kit includes a ready-to-use solution of the SYTOX Green nucleic acid stain. The SYTOX Green dye is impermeant to live cells and apoptotic cells but stains necrotic cells with intense green fluorescence by binding to cellular nucleic acids. After staining a cell population with Alexa Fluor 488 annexin V and SYTOX Green dye in the provided binding buffer, apoptotic cells show green fluorescence, dead cells show a higher level of green fluorescence and live cells show little or no fluorescence (Figure 15.5.7). These populations can easily be distinguished using a flow cytometer with the 488 nm spectral line of an argon-ion laser for excitation. Both Alexa Fluor 488 annexin and the SYTOX Green dye emit a green fluorescence that can be detected in the green channel, freeing the other channels for the detection of additional probes in multicolor labeling experiments. The kit components, number of assays and assay principles are summarized in Molecular Probes apoptosis assay kits—Table 15.4.

Figure 15.5.7 Flow cytometric analysis of Jurkat cells using the Single Channel Annexin V/Dead Cell Apoptosis Kit (V13240). Jurkat human T-cell leukemia cells were first exposed to 10 µM camptothecin for four hours green line) or left untreated (as control, blue line). Cells were then treated with the reagents in the kit and analyzed by flow cytometry. Note that the camptothecin-treated cells (green line) have a significantly higher percentage of apoptotic cells (intermediate green fluorescence) than the basal level of apoptosis seen in the control cells (blue line).

Alexa Fluor 488 Annexin V/Dead Cell Apoptosis Kit

Like the Single Channel Annexin V/Dead Cell Apoptosis Kit, our Alexa Fluor 488 Annexin V/Dead Cell Apoptosis Kit (V13241, V13245) detects the externalization of phosphatidylserine in apoptotic cells. This kit provides a sensitive two-color assay that employs our green-fluorescent Alexa Fluor 488 annexin and a ready-to-use solution of the red-fluorescent propidium iodide nucleic acid stain. Propidium iodide is impermeant to live cells and apoptotic cells but stains necrotic cells with red fluorescence, binding tightly to the nucleic acids in the cell. After staining a cell population with Alexa Fluor 488 annexin V and propidium iodide in the provided binding buffer, apoptotic cells show green fluorescence, dead cells show red and green fluorescence, and live cells show little or no fluorescence (Figure 15.5.8). These populations can easily be distinguished using a flow cytometer with the 488 nm spectral line of an argon-ion laser for excitation. The kit components, number of assays and assay principles are summarized in Molecular Probes apoptosis assay kits—Table 15.4.

Figure 15.5.8 Flow cytometric analysis of Jurkat cells using the Alexa Fluor 488 Annexin V/Dead Cell Apoptosis Kit (V13241, V13245). Jurkat human T-cell leukemia cells were first exposed to 10 µM camptothecin for four hours (right panel) or left untreated (as control, left panel). Cells were then treated with the reagents in the kit and analyzed by flow cytometry. Note that the camptothecin-treated cells have a significantly higher percentage of apoptotic cells (indicated by an "A") than the basal level of apoptosis seen in the control cells (top panel). V = viable cells, D = dead cells.

FITC Annexin V/Dead Cell Apoptosis Kit

FITC Annexin V/Dead Cell Apoptosis Kit (V13242) is similar to the Alexa Fluor 488 Annexin V/Dead Cell Apoptosis Kit, except that it contains fluorescein (FITC) annexin V in place of the Alexa Fluor 488 conjugate. The kit components, number of assays and assay principles are summarized in Molecular Probes apoptosis assay kits—Table 15.4.

Vybrant Apoptosis Assay Kit

The Vybrant Apoptosis Assay Kit #6 (V23200) is similar to the Alexa Fluor 488 Annexin V/Dead Cell Apoptosis Kit, except that it contains biotin-X annexin V and Alexa Fluor 350 streptavidin in place of the Alexa Fluor 488 conjugate. After staining a cell population with biotin-X annexin V in the provided binding buffer, Alexa Fluor 350 streptavidin is added to fluorescently label the bound annexin V. Finally, propidium iodide is added to detect necrotic cells. Apoptotic cells show blue fluorescence, dead cells show red and blue fluorescence and live cells show little or no fluorescence. These populations can easily be distinguished using a flow cytometer with UV excitation for the Alexa Fluor 350 fluorophore and 488 nm excitation for the propidium iodide. With this kit, fluorescence in the green channel is minimal. In the same experiment for apoptosis detection, the researcher can apply a green-fluorescent probe, for example an antibody labeled with the Alexa Fluor 488 dye or with fluorescein. The kit components, number of assays and assay principles are summarized in Molecular Probes apoptosis assay kits—Table 15.4.

PE Annexin V/ Dead Cell Apoptosis Kit and APC Annexin V/Dead Cell Apoptosis Kit

The PE Annexin V/ Dead Cell Apoptosis Kit and APC Annexin V/Dead Cell Apoptosis Kit (V35112, V35113) are  similar to the Single Channel Annexin V/Dead Cell Apoptosis Kit, except that they contain either R-phycoerythrin (R-PE) annexin V or allophycocyanin (APC) annexin V instead of Alexa Fluor 488 annexin V. In addition to the phycobiliprotein-conjugated annexin V, these kits include the SYTOX Green nucleic acid stain, which is impermeant to live cells and apoptotic cells but stains necrotic cells with intense green fluorescence. After staining a cell population with R-PE annexin V and SYTOX Green stain, apoptotic cells show orange fluorescence with very little green fluorescence, late apoptotic cells show a higher level of green and orange fluorescence and live cells show little or no fluorescence; these populations can easily be distinguished using a flow cytometer with the 488 nm spectral line of an argon-ion laser for excitation. After staining a cell population with APC annexin V and the SYTOX Green stain, apoptotic cells show far-red fluorescence with very little green fluorescence, late apoptotic cells show a higher level of green and far-red fluorescence and live cells show little or no fluorescence (Figure 15.5.9); these populations can easily be distinguished using a flow cytometer with both the 488 nm spectral line of an argon-ion laser and the 633 nm spectral line of a He-Ne laser for excitation. The kit components, number of assays and assay principles are summarized in Molecular Probes apoptosis assay kits—Table 15.4.

Figure 15.5.9 Flow cytometric analysis of Jurkat cells using the APC Annexin V/Dead Cell Apoptosis Kit (V35113). Jurkat human T-cell leukemia cells were first exposed to 10 µM camptothecin at 37°C, 5% CO2. The cells were then treated with the reagents in the kit and analyzed by flow cytometry. The SYTOX Green fluorescence versus allophycocyanin (APC) annexin fluorescence dot plot shows resolution of live, apoptotic and dead cell populations.

Metabolic Activity/Annexin V/Dead Cell Apoptosis Kit

The Metabolic Activity/Annexin V/Dead Cell Apoptosis Kit (V35114) is an enhanced version of the APC Annexin V/Dead Cell Apoptosis Kit. Nonfluorescent C₁₂-resazurin is reduced by viable cells to orange-fluorescent C₁₂-resorufin. After staining a cell population with allophycocyanin annexin V, C₁₂-resazurin and the SYTOX Green stain, apoptotic cells show far-red fluorescence, intermediate orange fluorescence and no green fluorescence; late apoptotic cells show intense far-red and green fluorescence and little orange fluorescence; live cells show little or no green or far-red fluorescence but significant orange fluorescence (Figure 15.5.10). The kit components, number of assays and assay principles are summarized in Molecular Probes apoptosis assay kits—Table 15.4.

Figure 15.5.10 Flow cytometric analysis of Jurkat cells using the Metabolic Activity/Annexin V/Dead Cell Apoptosis Kit (V35114). Jurkat human T-cell leukemia cells were first exposed to either 10 µM camptothecin or 2 mM hydrogen peroxide for 4 hours at 37°C, 5% CO₂. The cells were then combined, treated with the reagents in the kit and analyzed by flow cytometry. A) The SYTOX Green fluorescence versus allophycocyanin (APC) annexin fluorescence dot plot shows resolution of live, apoptotic and dead cell populations. The cell populations can be evaluated for metabolic activity using B) the dodecylresorufin fluorescence versus SYTOX Green fluorescence dot plot and C) the dodecylresorufin fluorescence versus allophycocyanin fluorescence dot plot.

Mitochondrial Membrane Potential/Annexin V Apoptosis Kit

The Mitochondrial Membrane Potential/Annexin V Apoptosis Kit (V35116) provides a rapid and convenient assay for two hallmarks of apoptosis—phosphatidylserine externalization and changes in mitochondrial membrane potential. Recombinant annexin V conjugated to the Alexa Fluor 488 dye, our brightest and most photostable green fluorophore, provides maximum sensitivity for detecting phosphatidylserine externalization in apoptotic cells. Live cells are labeled with MitoTracker Red CMXRos, which exhibits bright red fluorescence in the presence of a mitochondrial transmembrane potential. After staining a cell population with Alexa Fluor 488 annexin V and MitoTracker Red CMXRos dye in the provided binding buffer, live cells exhibit very little green fluorescence and bright red fluorescence, whereas apoptotic cells exhibit bright green fluorescence and decreased red fluorescence (Figure 15.5.11). These populations can easily be distinguished using a flow cytometer, and the 488 nm line of an argon-ion laser can be used to excite both dyes. The kit components, number of assays and assay principles are summarized in Molecular Probes apoptosis assay kits—Table 15.4.

Figure 15.5.11 Flow cytometric analysis of Jurkat cells using the Mitochondrial Membrane Potential/Annexin V Apoptosis Kit (V35116). Jurkat human T-cell leukemia cells in complete medium were first exposed to 10 µM camptothecin for 4 hours (left panel) or left untreated (right panel). Both cell populations were then treated with the reagents in the kit and analyzed by flow cytometry. Note that the apoptotic cells show higher reactivity for annexin V and lower MitoTracker Red dye fluorescence than do live cells.

Violet Chromatin Condensation/Dead Cell Apoptosis Kit

The Violet Chromatin Condensation/Dead Cell Apoptosis Kit (A35135) provides a rapid and convenient assay for apoptosis based upon fluorescence analysis of the compacted state of the chromatin in apoptotic cells. The kit contains the cell-permeant Vybrant DyeCycle Violet stain and the impermeant red-fluorescent SYTOX AADvanced dead cell stain. The condensed chromatin of apoptotic cells are stained more brightly by Vybrant DyeCycle Violet stain than the chromatin of normal cells. The SYTOX AADvanced stain labels only necrotic cells, based on membrane integrity. The staining pattern resulting from the simultaneous use of these stains makes it possible to distinguish normal, apoptotic, and necrotic cell populations by flow cytometry (Figure 15.5.12). The Vybrant DyeCycle Violet and SYTOX AADvanced stains are excited with the 405 nm violet diode laser and the 488 nm argon-ion laser, respectively.

Figure 15.5.12 Jurkat cells (human T-cell leukemia) treated with 10 μM camptothecin for 6 hours (panel B) or untreated (as control, panel A). Cells were then mixed with the reagents in the kit and analyzed by flow cytometry using 405/488 nm dual excitation. Note that the campthothecin-treated cells (panel B) have a higher percentage of apoptotic cells than the basal level of apoptosis seen in the control cells (panel A). A=apoptotic cells, V = viable cells, N = necrotic cells.

Violet Annexin V/Dead Cell Apoptosis Kit

Like the Single Channel Annexin V/Dead Cell Apoptosis Kit, the Violet Annexin V/Dead Cell Apoptosis Kit (A35136) detects the externalization of phosphatidylserine in apoptotic cells. This kit provides a sensitive two-color assay that employs our violet diode–excitable Pacific Blue annexin V (excitation/emission maxima ~415/460 nm) and the impermeant red-fluorescent SYTOX AADvanced dead cell stain. The SYTOX AADvanced stain labels only necrotic cells, based on membrane integrity. After staining a cell population with Pacific Blue annexin V and SYTOX AADvanced stain in the provided binding buffer, apoptotic cells show blue fluorescence, dead cells show red fluorescence, and live cells show little or no fluorescence. These populations can easily be distinguished with a flow cytometer equipped with both a 405 nm violet diode laser and an argon-ion laser for excitation. The kit components, number of assays and assay principles are summarized in Molecular Probes apoptosis assay kits—Table 15.4.

A distinctive feature of the early stages of apoptosis is the activation of caspase enzymes. Members of the caspase (CED-3/ICE) family of cysteine–aspartic acid specific proteases have been identified as crucial mediators of the complex biochemical events associated with apoptosis, The recognition site for caspases is marked by three to four amino acids followed by an aspartic acid residue, with the cleavage occurring after the aspartate. The caspase proteases are typically synthesized as inactive precursors. Inhibitor release or cofactor binding activates the caspase through cleavage at internal aspartates, either by autocatalysis or by the action of another protease. We offer a diverse selection of fluorogenic caspase substrates (Fluorogenic substrates for caspase activity—Table 15.5).

CellEvent Caspase-3/7 Green Detection Reagent

Caspase-3 (CPP32/apopain) is a key effector in the apoptosis pathway, amplifying the signal from initiator caspases (such as caspase-8) and signifying full commitment to cellular disassembly. In addition to cleaving other caspases in the enzyme cascade, caspase-3 has been shown to cleave poly(ADP-ribose) polymerase (PARP), DNA-dependent protein kinase, protein kinase Cδ and actin.

CellEvent Caspase-3/7 Green detection reagent (C10423) represents a new generation of caspase substrates and is an important tool for the study of apoptosis. The cell-permeant CellEvent reagent comprises the four–amino acid peptide DEVD—which contains the recognition site for caspases 3 and 7—conjugated to a nucleic acid–binding dye. Because the DEVD peptide inhibits the ability of the dye to bind to DNA, CellEvent Caspase-3/7 Green detection reagent is intrinsically nonfluorescent . In the presence of activated caspase 3/7, the dye is cleaved from the DEVD peptide and free to bind DNA, producing a bright green-fluorescent signal (absorption/emission maxima ~502/530 nm) indicative of apoptosis. This robust assay is highly specific for caspase 3/7 activation and, as expected, we observe nearly complete inhibition of the CellEvent Caspase-3/7 Green detection reagent signal in cells pretreated with a caspase 3/7 inhibitor.

Apoptosis assays with CellEvent Caspase-3/7 Green detection reagent are extremely easy to perform. Cells are simply incubated with the CellEvent reagent in complete culture medium for 30 minutes and then imaged by traditional fluorescence microscopy (Figure 15.5.13) or high-content imaging. Apoptotic cells with activated caspase 3/7 show bright green-fluorescent nuclei, whereas cells without activated caspase 3/7 show minimal fluorescence. Because the cleaved reagent labels nuclei of caspase 3/7–positive cells, this stain can also provide information on nuclear morphology, including condensed nuclei typical of late‑stage apoptosis.

One important advantage of the CellEvent caspase-3/7 assay is that no wash steps are required, thus preserving fragile apoptotic cells that are typically lost during these rinses. The loss of apoptotic cells during wash steps may lead to an underestimation of the extent of apoptosis in the sample, resulting in poor assay accuracy. Additionally, the fluorescent signal resulting from cleavage of CellEvent Caspase-3/7 detection reagent survives formaldehyde fixation and detergent permeabilization, providing the flexibility to perform endpoint assays and to probe for other proteins using immunocytochemical techniques.

Figure 15.5.13 Multiplex imaging of apoptosis. U2OS cells were treated with 30 μM etoposide for 18 hr to induce apoptosis. The treated cells were stained first with 7.5 μM CellEvent Caspase-3/7 Green detection reagent (green fluorescence, C10423) to detect apoptosis and Hoechst 33342 nucleic acid stain (blue fluorescence, H3570) to label nuclei, and then with 150 nM MitoTracker Deep Red FM (pink fluorescence, M22426) to label mitochondria. Following fixation and permeabilization, actin was labeled with Alexa Fluor 546 phalloidin (orange fluorescence, A22283).

Other Caspase 3 Substrates

The Z-DEVD-R110 substrate —a component of our EnzChek Caspase-3 Assay Kit #2 (E13184) and RediPlate 96 EnzChek Caspase-3 Assay Kit (R35100)—is available separately in a 20 mg unit size for high-throughput screening applications (R22120). This nonfluorescent bisamide is first converted by caspase-3 (or a closely related protease) to the fluorescent monoamide and then to the even more fluorescent rhodamine 110 (excitation/emission maxima ~496/520 nm). In addition, the bis-L-aspartic acid amide of R110 (D₂-R110, R22122), which contains rhodamine 110 (R110) flanked by aspartic acid residues, may serve as a substrate for a variety of apoptosis-related proteases, including caspase-3 and caspase-7, and does not appear to require any invasive techniques such as osmotic shock to gain entrance into the cytoplasm ().

Caspase-8 Substrates

Caspase-8 plays a critical role in the early cascade of apoptosis, acting as an initiator of the caspase activation cascade. Activation of the enzyme itself is accomplished through direct interaction with the death domains of cell-surface receptors for apoptosis-inducing ligands. The activated protease has been shown to be involved in a pathway that mediates the release of cytochrome c from the mitochondria and is also known to activate downstream caspases, such as caspase-3. A R110-based fluorogenic substrate containing the caspase-8 recognition sequence Ile-Glu-Thr-Asp (IETD) is available (Z-IETD-R110, R22125, R22126; Fluorogenic substrates for caspase activity—Table 15.5).

Other Caspase and Granzyme B Substrates

In addition to our R110-derived caspase-3 and -8 substrates, we offer R110-based substrates for caspase-1, -2, -6, -9 and -13, as well as for granzyme B (Fluorogenic substrates for caspase activity—Table 15.5). Granzyme B, a serine protease contained within cytotoxic T lymphocytes and natural killer cells, is thought to induce apoptosis in target cells by activating caspases and causing mitochondrial cytochrome c release.

EnzChek Caspase-3 Assay Kits

Molecular Probes EnzChek Caspase-3 Assay Kits permit the detection of apoptosis by assaying for increases in caspase-3 and caspase-3–like protease activities. Our EnzChek Caspase-3 Assay Kit #1 (E13183) contains the 7-amino-4-methylcoumarin (AMC)–derived substrate Z-DEVD-AMC () (where Z represents a benzyloxycarbonyl group). This substrate, which is weakly fluorescent in the UV spectral range (excitation/emission maxima ~330/390 nm), yields the blue–fluorescent product AMC (A191, Introduction to Enzyme Substrates and Their Reference Standards—Section 10.1, ), which has excitation/emission maxima of 342/441 nm upon proteolytic cleavage.

The EnzChek Caspase-3 Assay Kit #2 (E13184) contains the R110-derived substrate, Z-DEVD-R110 (). This substrate is a bisamide derivative of R110, containing DEVD peptides covalently linked to each of R110's amino groups, thereby suppressing both the dye's visible absorption and fluorescence. Upon enzymatic cleavage by caspase-3 (or a closely related protease), the nonfluorescent bisamide substrate is converted in a two-step process first to the fluorescent monoamide and then to the even more fluorescent R110 (R6479, Introduction to Enzyme Substrates and Their Reference Standards—Section 10.1, ). Both of these hydrolysis products exhibit spectral properties similar to those of fluorescein, with excitation/emission maxima of 496/520 nm. The Z-DEVD-R110 substrate (R22120) is also available separately in a 20 mg unit size for high-throughput screening applications.

Either kit can be used to continuously measure the activity of caspase-3 and closely related proteases in cell extracts and purified enzyme preparations using a fluorescence microplate reader or fluorometer. AMC-based DEVD substrates, which yield blue fluorescence upon proteolytic cleavage, are widely used to monitor caspase-3 activity. The longer-wavelength spectra and higher extinction coefficient of the green-fluorescent products of the R110-based substrate in Kit #2 (E13184) should provide even greater sensitivity. The reversible aldehyde-based inhibitor Ac-DEVD-CHO can be used to confirm that the observed fluorescence signal in both induced and control cell populations is due to the activity of caspase-3–like proteases. The EnzChek Caspase-3 Assay Kits contain:

• Z-DEVD-AMC (in Kit #1, E13183) or Z-DEVD-R110 (in Kit #2, E13184)
• Dimethylsulfoxide (DMSO)
• Concentrated cell-lysis buffer
• Concentrated reaction buffer
• Dithiothreitol (DTT)
• Ac-DEVD-CHO, a reversible aldehyde-based inhibitor
• 7-Amino-4-methylcoumarin (AMC) (in Kit E13183) or rhodamine 110 (in Kit E13184) reference standard to quantitate the amount of fluorophore released in the reaction
• Detailed protocols (EnzChek Caspase-3 Assay Kit #1 *Z-DEVD-AMC Substrate*, EnzChek Caspase-3 Assay Kit #2 *Z-DEVD-R110 Substrate*)

Each kit provides sufficient reagents for performing ~500 assays using a volume of 100 µL per assay.

RediPlate 96 EnzChek Caspase-3 Assay Kit

Our EnzChek Caspase-3 Assay Kit #2 is also available as a convenient RediPlate 96 EnzChek Caspase-3 Assay Kit (R35100), which includes one 96-well microplate, contained in a resealable foil packet to ensure the integrity of the fluorogenic components, plus all necessary buffers and reagents for performing the assay. The enzyme sample to be assayed is added to the microplate in a suitable buffer, along with any compounds to be tested. Then, after incubation, the resultant fluorescence is quantitated on a fluorescence microplate reader equipped with filters appropriate for the green-fluorescent R110, with excitation/emission maxima of 496/520 nm. The microplate consists of twelve removable strips, each with eight wells, allowing researchers to perform only as many assay as required for the experiment. Eleven of the strips (88 wells) are preloaded with the Z-DEVD-R110 substrate. The remaining strip, marked with blackened tabs, contains a dilution series of free R110 that may be used as a fluorescence reference standard. The reversible aldehyde-based inhibitor Ac-DEVD-CHO, which is supplied in a separate vial, can be used to confirm that the observed fluorescence signal in both induced and control cell populations is due to the activity of caspase-3–like proteases. RediPlate Assay Kits—Table 10.3 summarizes our other RediPlate 96 and RediPlate 384 Assay Kits for protease activity (Detecting Peptidases and Proteases—Section 10.4), phosphatase activity (Detecting Enzymes That Metabolize Phosphates and Polyphosphates—Section 10.3) and RNA quantitation (Nucleic Acid Detection and Quantitation in Solution—Section 8.3).

Image-iT LIVE Green Caspase Detection Kits for Fluorescence Microscopy

The Image-iT LIVE Green Caspase-3 and -7 Detection Kit, Image-iT LIVE Green Caspase-8 Detection Kit and Image-iT LIVE Green Poly Caspases Detection Kit (I35106, I35105, I35104) employ a novel approach to detect active caspases that is based on a fluorescent inhibitor of caspases (FLICA methodology). The FLICA inhibitor comprises a fluoromethyl ketone (FMK) moiety, which can react covalently with a cysteine, a caspase-selective amino acid sequence and a fluorescent carboxyfluorescein (FAM) reporter group. Essentially an affinity label, the FLICA inhibitor is thought to interact with the enzymatic reactive center of an activated caspase via the recognition sequence, and then to attach covalently to a cysteine through the reactive FMK moiety. The FLICA inhibitor's recognition sequence is aspartic acid–glutamic acid–valine–aspartic acid (DEVD) for caspase-3 and-7 detection, leucine–glutamic acid–threonine–aspartic acid (LETD) for caspase-8 detection and valine–alanine–aspartic acid (VAD) for detection of most caspases (including caspase-1, -3, -4, -5, -6, -7, -8 and -9). Importantly, the FLICA inhibitor is cell permeant and not cytotoxic; unbound FLICA molecules diffuse out of the cell and are washed away. The remaining green-fluorescent signal (excitation/emission maxima ~488/530 nm) can be used as a direct measure of the amount of active caspase that was present at the time the inhibitor was added. FLICA reagents have been used widely to study apoptosis with flow cytometry and microscopy. Recent work indicates that cellular fluorescence from the bound FLICA reagent is strongly linked to caspase activity in apoptotic cells; however, the interaction of the FLICA reagent with other cellular sites may contribute to signal intensity in nonapoptotic cells. The Image-iT LIVE Green Caspase Detection Kit includes:

• FAM-DEVD-FMK caspase-3 and -7 reagent (in Kit I35106), FAM-LETD-FMK caspase-8 reagent (in Kit I35105) or FAM-VAD-FMK poly caspases reagent (in Kit I35104)
• Hoechst 33342
• Propidium iodide
• Dimethylsulfoxide (DMSO)
• Apoptosis fixative solution
• Concentrated apoptosis wash buffer
• Detailed protocols for fluorescence microcscopy assays (Image-iT LIVE Green Caspase Detection Kits)

In addition to a specific FLICA reagent, each kit provides Hoechst 33342 and propidium iodide stains, which allow the simultaneous evaluation of caspase activation, nuclear morphology and plasma membrane integrity. Sufficient reagents are provided for 25 assays, based on labeling volumes of 300 µL. These Image-iT LIVE Green Caspase Detection Kits can also be used in combination with other reagents for multiparametric study of apoptosis.

Image-iT LIVE Red Caspase Detection Kits for Fluorescence Microscopy

The Image-iT LIVE Red Caspase-3 and -7 Detection Kit and Image-iT LIVE Red Poly Caspases Detection Kit (I35102, I35101) are analogous to the Image-iT LIVE Green Caspase Detection Kits except that the FLICA reagent contains a red-fluorescent sulforhodamine (SR) reporter group instead of a green-fluorescent carboxyfluorescein (FAM) reporter group. This assay's red-fluorescent signal (excitation/emission maxima ~550/595 nm) can be used as a direct measure of the amount of active caspase that was present at the time the inhibitor was added. The Image-iT LIVE Red Caspase Detection Kit includes:

• SR-DEVD-FMK caspase-3 and -7 reagent (in Kit I35102) or SR-VAD-FMK poly caspases reagent (in Kit I35101)
• Hoechst 33342
• SYTOX Green nucleic acid stain
• Dimethylsulfoxide (DMSO)
• Apoptosis fixative solution
• Concentrated apoptosis wash buffer
• Detailed protocols for fluorescence microcscopy assays (Image-iT LIVE Red Caspase Detection Kits) 

In addition to a specific FLICA reagent, each kit provides Hoechst 33342 and SYTOX Green nucleic acid stains, which allow the simultaneous evaluation of caspase activation, nuclear morphology and plasma membrane integrity. Sufficient reagents are provided for 25 assays, based on labeling volumes of 300 µL.

Vybrant FAM Caspase Assay Kits for Flow Cytometry

Like the Image-iT Kits described above, the Vybrant FAM Caspase Assay Kits for flow cytometry are based on a fluorescent caspase inhibitor (FLICA methodology). We offer three different Vybrant FAM Caspase Assay Kits designed to target different caspases. The Vybrant FAM Caspase-3 and -7 Assay Kit (V35118) provides a FLICA inhibitor containing the caspase-3 and -7 recognition sequence DEVD; the Vybrant FAM Caspase-8 Assay Kit (V35119) provides a FLICA inhibitor containing the caspase-8 recognition sequence Leu-Glu-Thr-Asp (LETD); and the Vybrant FAM Poly Caspases Assay Kit (V35117) provides a FLICA inhibitor containing the caspase recognition sequence Val-Ala-Asp (VAD), which is recognized by caspase-1, -3, -4, -5, -6, -7, -8 and -9. In addition to the selective FLICA reagent, these kits contain the Hoechst 33342 and propidium iodide nucleic acid stains to permit simultaneous evaluation of caspase activation, membrane permeability and cell cycle. The Vybrant FAM Caspase Assay Kits include:

• FAM-DEVD-FMK caspase-3 and -7 reagent (in Kit V35118), FAM-LETD-FMK caspase-8 reagent (in Kit V35119) or FAM-VAD-FMK poly caspases reagent (in Kit V35117)
• Hoechst 33342
• Propidium iodide
• Dimethylsulfoxide (DMSO)
• Apoptosis fixative solution
• Concentrated apoptosis wash buffer
• Detailed protocols for flow cytometry assays (Vybrant FAM Caspase-3 and -7 Assay Kit, Vybrant FAM Caspase-8 Assay Kit, Vybrant FAM Poly Caspases Assay Kit)

Sufficient reagents are provided for 25 assays, based on labeling volumes of 300 µL. These Vybrant FAM Caspase Assay Kits can be used in combination with other fluorescent probes, such as the far-red–fluorescent allophycocyanin annexin V (A35110), for a multiparameter study of apoptosis.

Cathepsins and Calpains

The role of intracellular cathepsins and calpains in apoptosis is unclear, although an upstream role of cathepsin B in activation of some caspases and cathepsins during apoptosis has been established. Pepstatin A, which is a selective inhibitor of carboxyl (acid) proteases such as cathepsin D, has been reported to inhibit apoptosis in microglia, lymphoid cells and HeLa cells. Consequently, our cell-permeant BODIPY FL pepstatin derivative (P12271), which we have shown to inhibit cathepsin D in vitro (IC₅₀ ~10 nM) and to target cathepsin D within lysosomes of live and fixed cells, has demonstrable utility for following the intracellular translocation of cathepsin D.

Calpains are a family of ubiquitous calcium-activated thiol proteases that are implicated in a variety of cellular functions including exocytosis, cell fusion, apoptosis and cell proliferation. Caspase-dependent downstream processing of calpain has been reported, suggesting that calpain may play a role in the degradation phase of apoptosis that is distinct from that of caspases. One mechanism of caspase dependence appears to be processing of the endogenous calpain inhibitor calpastin by caspases. However, calpain activation has also been reported to be upstream of caspases in radiation-induced apoptosis. Our t-BOC-Leu-Met-CMAC fluorogenic substrate (A6520) has been used to measure calpain activity in hepatocytes following the addition of extracellular ATP and may be of utility in detecting caspase-activated processing of procalpain in live single cells. Peptidase substrates based on our CMAC fluorophore (7-amino-4-chloromethylcoumarin, C2110; Introduction to Enzyme Substrates and Their Reference Standards—Section 10.1) passively diffuse into several types of cells, where the thiol-reactive chloromethyl group is enzymatically conjugated to glutathione by intracellular glutathione S-transferase or reacts with protein thiols, thus transforming the substrate into a membrane-impermeant probe. Subsequent peptidase cleavage results in a bright blue-fluorescent glutathione conjugate; see Detecting Peptidases and Proteases—Section 10.4 for more information on AMC- and CMAC-based peptidase substrates.

A distinctive feature of the early stages of programmed cell death is the disruption of active mitochondria. This mitochondrial disruption includes changes in the membrane potential and alterations to the oxidation–reduction potential of the mitochondria. Changes in the membrane potential are presumed to be due to the opening of the mitochondrial permeability transition pore, allowing passage of ions and small molecules. The resulting equilibration of ions leads in turn to the decoupling of the respiratory chain and then the release of cytochrome c into the cytosol. These changes can be monitored using our extensive selection of potential-sensitive mitochondrial stains (Probes for Mitochondria—Section 12.2). We also offer several kits providing ready-to-use formulations of these reagents in flow cytometry or imaging protocols.

Image-iT LIVE Mitochondrial Transition Pore Assay Kit for Fluorescence Microscopy

The Image-iT LIVE Mitochondrial Transition Pore Assay Kit (I35103), based on published experimentation for mitochondrial transition pore opening, provides a more direct method of measuring mitochondrial permeability transition pore opening than assays relying on mitochondrial membrane potential alone. This assay employs the acetoxymethyl (AM) ester of calcein, a colorless and nonfluorescent esterase substrate, and CoCl₂, a quencher of calcein fluorescence, to selectively label mitochondria. Cells are loaded with calcein AM, which passively diffuses into the cells and accumulates in cytosolic compartments, including the mitochondria. Once inside cells, calcein AM is cleaved by intracellular esterases to liberate the very polar fluorescent dye calcein, which does not cross the mitochondrial or plasma membranes in appreciable amounts over relatively short periods of time. The fluorescence from cytosolic calcein is quenched by the addition of CoCl₂, while the fluorescence from the mitochondrial calcein is maintained. As a control, cells that have been loaded with calcein AM and CoCl₂ can also be treated with a Ca²⁺ ionophore such as ionomycin (I24222, Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents—Section 19.8) to allow entry of excess Ca²⁺ into the cells, which triggers mitochondrial pore activation and subsequent loss of mitochondrial calcein fluorescence. This ionomycin response can be blocked with cyclosporine A, a compound reported to prevent mitochondrial transition pore formation by binding cyclophilin D.

The Image-iT LIVE Mitochondrial Transition Pore Assay Kit has been tested with HeLa cells and bovine pulmonary artery endothelial cells (BPAEC). Each Image-iT LIVE Mitochondrial Transition Pore Assay Kit provides:

• Calcein AM
• MitoTracker Red CMXRos, a red-fluorescent mitochondrial stain (excitation/emission maxima ~579/599 nm)
• Hoechst 33342, a blue-fluorescent nuclear stain (excitation/emission maxima ~350/461 nm)
• Ionomycin
• CoCl₂
• Dimethylsulfoxide (DMSO)
• Detailed protocols (Image-iT LIVE Mitochondrial Transition Pore Assay Kit)

Sufficient reagents are provided for 100 assays, based on labeling volumes of 1 mL.

MitoProbe Transition Pore Assay Kit for Flow Cytometry

The MitoProbe Transition Pore Assay Kit (M34153), based on published experimentation for mitochondrial transition pore opening, provides a more direct method of measuring mitochondrial permeability transition pore opening than assays relying on mitochondrial membrane potential alone. As with the Image-iT LIVE mitochondrial transition pore assay described above, this assay employs the acetoxymethyl (AM) ester of calcein, a colorless and nonfluorescent esterase substrate, and CoCl₂, a quencher of calcein fluorescence, to selectively label mitochondria. Cells are loaded with calcein AM, which passively diffuses into the cells and accumulates in cytosolic compartments, including the mitochondria. Once inside cells, calcein AM is cleaved by intracellular esterases to liberate the very polar fluorescent dye calcein, which does not cross the mitochondrial or plasma membranes in appreciable amounts over relatively short periods of time. The fluorescence from cytosolic calcein is quenched by the addition of CoCl₂, while the fluorescence from the mitochondrial calcein is maintained. As a control, cells that have been loaded with calcein AM and CoCl₂ can also be treated with a Ca²⁺ ionophore such as ionomycin (I24222, Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents—Section 19.8) to allow entry of excess Ca²⁺ into the cells, which triggers mitochondrial pore activation and subsequent loss of mitochondrial calcein fluorescence. This ionomycin response can be blocked with cyclosporine A, a compound reported to prevent mitochondrial transition pore formation by binding cyclophilin D.

The MitoProbe Transition Pore Assay Kit has been tested with Jurkat cells, MH1C1 cells and bovine pulmonary artery endothelial cells (BPAEC). Each MitoProbe Transition Pore Assay Kit provides:

• Calcein AM
• CoCl₂
• Ionomycin
• Dimethylsulfoxide (DMSO)
• Detailed protocols (MitoProbe Transition Pore Assay Kit)

Sufficient reagents are provided for 100 assays, based on labeling volumes of 1 mL.

MitoProbe JC-1 Assay Kit for Flow Cytometry

The MitoProbe JC-1 Assay Kit (M34152) provides the cationic dye JC-1 and a mitochondrial membrane potential uncoupler, CCCP (carbonyl cyanide 3-chlorophenylhydrazone), for the study of mitochondrial membrane potential. JC-1 () exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (~529 nm) to red (~590 nm), due to concentration-dependent formation of red-fluorescent J-aggregates. Consequently, mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio, which is dependent only on the membrane potential and not on other factors such as mitochondrial size, shape and density, which may influence single-component fluorescence measurements. Use of fluorescence ratio detection therefore allows researchers to make comparative measurements of membrane potential and to determine the percentage of mitochondria within a population that respond to an applied stimulus. Subtle heterogeneity in cellular responses can be discerned in this way. For example, four distinct patterns of mitochondrial membrane potential change in response to glutamate receptor activation in neurons have been identified using confocal ratio imaging of JC-1 fluorescence.

JC-1 can be used as an indicator of mitochondrial potential in a variety of cell types, including myocytes and neurons, as well as in intact tissues and isolated mitochondria. JC-1 is more specific for mitochondrial versus plasma membrane potential and more consistent in its response to depolarization than some other cationic dyes such as DiOC₆(3) and rhodamine 123. The most widely implemented application for JC-1 is the detection of mitochondrial depolarization occurring in apoptosis.  Each MitoProbe JC-1 Assay Kit provides:

• JC-1
• Dimethylsulfoxide (DMSO)
• CCCP
• Concentrated phosphate-buffered saline (PBS)
• Detailed protocols (MitoProbe JC-1 Assay Kit for Flow Cytometry)

Sufficient reagents are provided for 100 assays, based on a labeling volume of 1 mL.

MitoProbe DiIC₁(5) and MitoProbe DiOC₂(3) Assay Kits for Flow Cytometry

Cationic carbocyanine dyes have been shown to accumulate in cells in response to membrane potential, and membrane potential changes have been studied in association with apoptosis. The MitoProbe DiIC₁(5) and MitoProbe DiOC₂(3) Assay Kits (M34151, M34150) provide solutions of the far-red–fluorescent DiIC₁(5) (1,1',3,3,3',3'-hexamethylindodicarbocyanine iodide) and green-fluorescent DiOC₂(3) (3,3'-diethyloxacarbocyanine iodide) carbocyanine dyes, respectively, along with a mitochondrial membrane potential disrupter, CCCP, for the study of mitochondrial membrane potential. These DiIC₁(5) and DiOC₂(3) carbocyanine dyes penetrate the cytosol of eukaryotic cells and, at concentrations below 100 nM, accumulate primarily in mitochondria with active membrane potentials. In the case of DiOC₂(3), this accumulation is accompanied by a shift from green to red emission due to dye stacking, allowing the use of a ratiometric parameter (red/green fluorescence ratio) that corrects for size differences when measuring membrane potential in bacteria. DiIC₁(5) and DiOC₂(3) stain intensities decrease when cells are treated with reagents that disrupt mitochondrial membrane potential, such as CCCP. Each MitoProbe DiIC₁(5) and MitoProbe DiOC₂(3) Assay Kit provides:

• DiIC₁(5) (in Kit M34151) or DiOC₂(3) (in Kit M34150)
• CCCP
• Detailed protocols for labeling cells with the short-chain carbocyanine dye, as well as with annexin V conjugates (not included) (MitoProbe DiIC1(5) Assay Kit for Flow Cytometry, MitoProbe DiOC2(3) Assay Kit for Flow Cytometry)

Cells stained with DiIC₁(5) can be visualized by flow cytometry with red excitation and far-red emission filters; cells stained with DiOC₂(3) can be visualized by flow cytometry with blue excitation and green and red emission filters. DiIC₁(5) can be paired with other reagents, such as propidium iodide and the green-fluorescent Alexa Fluor 488 annexin V (both provided in the Alexa Fluor 488 Annexin V/Dead Cell Apoptosis Kit; V13241, V13245), for multiparameter study of vitality and apoptosis. DiOC₂(3) can be paired with other reagents, such as the far-red–fluorescent allophycocyanin annexin V (A35110), for multiparameter study of vitality and apoptosis. Combining these short-chain carbocyanine dyes with annexin V conjugates results in superior resolution of subpopulations when compared with results obtained with other commonly used dyes.

Autophagy describes the segregation and delivery of cytoplasmic cargo, including proteins and organelles, for degradation by hydrolytic enzymes. The process of autophagy begins with the formation and elongation of isolation membranes, or phagophores (Figure 15.5.14). The cytoplasmic cargo is then sequestered, and the double-membrane autophagosome fuses with a lysosome to generate the autolysosome. Finally, degradation is achieved through the action of hydrolytic enzymes within the autolysosome.

Autophagy was first described in 1963; however, only in the past decade has this pathway become the subject of intense study. Researchers have sought to gain further insight into the role basal autophagy plays in cell homeostasis and development, and to further elucidate the role of induced autophagy in the cell’s response to stress, microbial infection and disease.

Figure 15.5.14 Schematic depiction of the multistep autophagy pathway in a eukaryotic cell. The first step involves the formation and elongation of isolation membranes, or phagophores. In the second step, which involves the LC3B protein, the cytoplasmic cargo is sequestered, and the double-membrane autophagosome is formed. Fusion of a lysosome with the autophagosome to generate the autolysosome is the penultimate step. In the fourth and final phase, the cargo is degraded.

Autophagy Analysis Through Observation of LC3B

The LC3 protein plays a critical role in autophagy. Normally this protein resides in the cytosol, but following cleavage and lipidation with phosphatidylethanolamine, LC3 associates with the phagophore and can be used as a general marker for autophagic membranes (Figure 15.5.14). The new Premo Autophagy Sensor Kits (P36235, P36236) combine the selectivity of an LC3B–fluorescent protein chimera with the transduction efficiency of BacMam technology (BacMam Gene Delivery and Expression Technology—Note 11.1), enabling unambiguous visualization of this protein in live cells (Figure 15.5.15). Recent improvements made to the BacMam system enable efficient transduction in a wider variety of cells, including neurons and neural stem cells (NSCs) with an easy, one-step protocol. To image autophagy, BacMam LC3B-FP is simply added to cells and allowed to incubate overnight for protein expression. Each Premo Autophagy Sensor Kit includes:

• BacMam LC3B-FP (GFP fusion, P36235; RFP fusion, P36236)
• Control BacMam LC3B (G120A)-FP
• Chloroquine diphosphate, to artificially induce phagosome formation
• Detailed protocols (Premo Autophagy Sensors)

Following treatment with chloroquine diphosphate, normal autophagic flux is disrupted, and autophagosomes accumulate as a result of the increase in lysosomal pH. The mutation in the control BacMam LC3B (G120A)-FP prevents cleavage and subsequent lipidation during normal autophagy, and thus protein localization should remain cytosolic and diffuse.

In addition to the Premo Autophagy Sensor Kits, we offer the LC3B Antibody Kit for Autophagy (L10382), which includes a rabbit polyclonal anti-LC3B antibody and chloroquine diphosphate for imaging autophagy via LC3B localization in fixed samples. The LC3B Antibody for Kit Autophagy has been validated for use with fluorescence microscopy and high-content imaging and analysis.

Figure 15.5.15 Detecting autophagy with the Premo Autophagy Sensor and fluorescence microscopy (A) or high-content imaging and analysis (B). (A) U2OS cells were cotransduced with the Premo Autophagy Sensor LC3B-RFP (P36236) and CellLight MAP4-GFP (C10598). The following day, cells were treated with 50 μM chloroquine. The following day, cells were incubated with 1 μg/mL Hoechst 33342 before imaging. (B) HeLa cells were plated at 5000 cells per well and left to adhere overnight. Cells were then transduced with the Premo Autophagy Sensor LC3B-GFP. The following day, cells were incubated with 50 μM chloroquine or left untreated (control) for 16 hr. Quantitative analysis was performed by quantifying fluorescence from vesicular structures in the perinuclear region using the Thermo Scientific Cellomics ArrayScan VTI platform.

Imaging Autophagic Organelles

Two organelles that play a crucial role in autophagy are the mitochondria and lysosomes. Old, damaged, or surplus mitochondria are a major target for autophagy, which in this case is sometimes referred to as "mitophagy." Degradation of mitochondria through this process can be used to recover their amino acids and other nutrients, as well as to remove damaged mitochondria from the cell. Fusion of a lysosome with the phagophore to form the autolysosome is the penultimate step of the autophagic pathway (Figure 15.5.13). A variety of reagents including fluorescent dyes, fluorescent protein (FP) chimeras and antibodies can be used to image mitochondria and lysosomes during basal and induced autophagy (Fluorescent detection reagents for imaging mitochondria and lysosomes—Table 15.6). For a description of our mitochondria and lysosomal-selective organelle probes, see Probes for Mitochondria—Section 12.2 and Probes for Lysosomes, Peroxisomes and Yeast Vacuoles—Section 12.3.

Visualizing Autolysosome Formation

In conjunction with the Premo autophagy sensors LC3B-GFP and LC3B-RFP, the fluorogenic protease substrates DQ Green BSA and DQ Red BSA can be used to accurately image the formation of the autolysosome in live cells. DQ Green BSA and DQ Red BSA (D12050, D12051; Detecting Peptidases and Proteases—Section 10.4) are bovine serum albumin (BSA) conjugates that have been labeled to such a high degree that the fluorescence is self-quenched. To visualize autolysosome formation, cells that express a GFP- or RFP-LC3 are incubated with the contrasting color of DQ BSA. The convergence of the lysosome with the autophagosome results in dequenching and release of brightly fluorescent fragments. The autolysosomes can then be identified by co-localization of green and red fluorescence.