How to measure early apoptotic events?

Apoptosis and cellular apoptotic events. Source: tebu-bio

Fig. 1: Process of Apoptosis

Apoptosis is the most prominent process of programmed cell death (for an overview see Fig. 1). Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation. Furthermore, changes affecting the membrane, nucleus, cytoplasm, and mitochondria occur. Apoptosis involves a complex cascade of reactions regulated by specific proteases called caspases (take a look at previous posts on Caspases as pharmaceutical targets – how to screen for inhibitors?), and results in DNA degradation. Apoptotic processes have been researched in an extensive variety of diseases. Excessive apoptosis causes atrophy, whereas an insufficient amount results in uncontrolled cell proliferation, such as cancer.

Besides apoptosis other types of programed cell death are known, such as autophagy (see How to manipulate and measure Autophagy?), necroptosis, and ferroptosis (look out for an imminent post I’ll be doing about this iron-dependent form of cell death very shortly, as well as tools to differentiate between apoptois, necroptosis, autophagy, and ferroptosis).

In this post, let’s take a look at methods and kits allowing to measure early apoptotic events.

Methods to measure early apoptosis

The onset of the execution phase of apoptosis can be linked to translocation of phosphatidylserine from the interior to the exterior surface of the mammalian cell membrane.

Apopercentage principle - tebu-bio

Fig. 2: Apoptose related translocation of phosphatidylserine to the outer leaflet of the cell membrane and principle of the Apopercentags assay.

Two different assay types are available to measure this process.

Direct measurement of translocation of phosphatidylserine

Choline phospholipids make up most of the lipid in the extracellular membrane.

This organisational composition is essential for the normal functions of a viable cell, including the insertion of protein receptors and transporters between the phospholipid molecules. Maintaining the asymmetric composition is an energy dependant process involving the activity of enzymes, termed “flippases”.

In apoptotic committed cells flippase regulation is either overwhelmed, or is inactivated by the activity of the enzyme “scramblase” (floppase) (see Fig 2).

Apopercentage graph - tebu-bio.

Fig. 3: Effect of Hydrogen Peroxide (0–10mM) for 4 hours on CHO Cells. Colorimetric quantification.

 

 

 

 

 

 

 

 

 

The Apopercentageassay kit utilizes the fact that in early apoptotic cells, phosphatidylserine appears in the outer leaflet of the cell membrane thus allowing a specific dye (APOP% dye) to bind to and enter the cell (Fig. 2).

The uptake of the dye is uni-directional, leading to dye accumulation within the cell. The dye accumulation can be finally measured with a microplate colorimeter (Fig. 3) and followed with a microscope (Fig. 4).

Apopercentage -microscopic measurement - tebu-bio

Fig. 4: Incubation of 3T3 cells with 5 mMHydrogen Peroxide for different periods.

Measurement of Annexin V binding to apoptotic cells

Annexin V based apoptosis assays are based on the same principle as the Apopercentage kit described above.

Phosphatidylserine appears on the surface of early apoptotic cells, and as Annexin V has a high affinity for this specific phospholipid. It can be easily detected by staining for instance with a fluorescent conjugate of Annexin V or by biotinylated Annexin V and subsequent detection by conventional dye-staining using any streptavidine- or avidin-dye reagents (Fig. 5).

Apoptosis related cell based caspase assays

Annexin V assay principle - tebu-bio

Fig. 5: Principle of the Annexin V based Apoptosis assay

Several caspases are involved in initiation and downstream processes of apoptosis (others play a critical role as mediators of innate immune responses).

Apoptose related caspases can be divided into Initiator caspases (caspase-8, and -9) and executioner caspases (caspase-3, -6, and -7).

Initiator caspases are activated by dimerization of  inactive procaspase monomers, while executioner Caspases have to be cleaved and thus activated by initiator caspases.

 

Executioner caspase exists as procaspase dimers which upon cleavage and subsequent conformational change results in the mature and active enzyme.

The activated executioner caspases finally lead to destruction of key structural proteins and activate other enzymes.

Thus in-cell caspase activation can be used as an indicator of the initiation of the apoptotic process. Furthermore, assays measuring the in-cell activation of caspases can be used to distinguish apoptosis from necrosis.

You’ll find the range of kits (see exemplary results in Fig. 6) for whole cell detection of caspase activity in apoptotic cells on the following links:

  • Initiator Caspases 8 and 9

    Poly-caspase detection kit - tebu-bio.

    Fig. 6. Jurkat cells were either treated with DMSO (negative, non-induced cells – left) or with staurosporine (apoptotic, induced cells – right) for 2 hours at 37°C. Cells were labeled with FAM-VAD-FMK (detection compound used in the poly caspase detection kit) solution for 60 minutes at 37°C and read on a 96-well fluorescence plate reader. As the caspases became more active, indicating apoptosis, the amount of green fluorescence increased by over 500% in the induced Jurkat cells.

  • Executioner Caspases 3, 6, and 7

In one of my next posts,  I’ll go into these cell based caspase assays in more detail.

 Assays to measure the disruption of the mitochondrial transmembrane potential

Disruption of the mitochondrial transmembrane potential is another early intracellular event that occurs following induction of apoptosis. To distinguish between healthy and apoptotic cells, changes in the mitochondrial transmembrane potential can be measured.
In assays based on the mitochondrial transmembrane potential cationic dyes that fluoresce differently in healthy versus apoptotic cells are used. In healthy cells, the dyes accumulate and aggregate in the mitocondria (red fluorescence), while in apoptotic cells, they cannot aggregate in the mitochondria due to the altered mitochondrial transmembrane potential, and thus remain in the cytoplasm (green fluorescence).

Assay to measure Cytochrome c release from mitochondria to the cytosol

Cytochrome c plays an important role in apoptosis. The protein is located in the space between the inner and outer mitochondrial membranes. An apoptotic stimulus triggers the release of cytochrome c from the mitochondria into cytosol where it binds to Apaf-1. The cytochrome c/Apaf-1 complex activates caspase-9, which then activates caspase-3 and other downstream caspases.
The Cytochrome c Releasing Apoptosis Assay Kit provides an effective means for detecting cytochrome c translocation from mitochondria into cytosol during apoptosis. The principle of the kit is based on the isolation of a highly enriched mitochondria fraction from cytosol without any ultracentrifugation step. Cytochrome c releasing from mitochondria into cytosol is then determined by Western blotting using the cytochrome c antibody provided in the kit.

Multi-Parameter Apoptosis Assay Kit

Multi parameter assay

Fig. 7: Hoechst staining shows that most control cells have round and intact nuclei (Panel A) whereas staurosporine-treated cells mostly have condensed and fragmented nuclei (Panel D). TMRE staining reveals that most control cells have undisrupted mitochondrial membrane potential (Panel B) whereas staurosporine-treated cells mostly have diminished membrane potential and were not stained (Panel E). Most control cells are Annexin V negative (Panel C) whereas cells treated with staurosporine are mostly Annexin V positive (Panel F), indicating cells are undergoing apoptosis.

And finally, there is a kit combining the strong points of divers methods to detect early apoptosis.

The Multi-Parameter Apoptosis Assay Kit employs FITC-conjugated Annexin V as a probe for phosphatidylserine on the outer membrane of apoptotic cells, TMRE as a probe for mitochondrial membrane potential, 7-AAD as an indicator of membrane permeability/cell viability, and Hoechst Dye to demonstrate nuclear morphology. The kit allows phenotypic characterization of different cell death parameters at a single-cell level.

 The example (Fig. 7) showns an experiment with Staurosporine which induces apoptosis in Jurkat cells, as measured by nuclear morphology, a decrease in mitochondrial membrane potential, and an increase of Annexin V FITC positive cells. Panels A-C show same field of cells from the control group whereas panels D-F show the same field of cells from the staurosporine-treated group.

Just a little bit more information I would like to share:
Any questions concerning the methods and kits presented in this post? Contact me through the form below!

Trackbacks

  1. […] the most prominent mode of programmed cell death – Apoptosis. In a recent post, I summarized methods to detect early Apoptosis and at the end of this post you’ll find a list of apoptosis modulators (available through […]

  2. […] induction of apoptosis leads to characteristic cell changes and finally to death (see How to measure early apoptotic events). These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, […]

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