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Studying cell death

Distinguishing between different forms of non-apoptotic cell death can be challenging, especially as many share similar morphological features (Table 1). However, the distinct regulatory pathways involved with each provide distinct protein markers that can be used for detection.

Table 1. Summary of main cell death pathways

Whatever mode of cell death you are studying, a combination of different approaches should be used. Study of non-apoptotic cell death should use both specific positive indicators of the cell death mode of interest coupled with cell viability assays and techniques to rule out apoptosis²⁸.

Cell viability

Cell viability can be assessed using parameters such as cytolysis, metabolic activity or senescence. Your choice of assay will depend on what you want to detect, what instrumentation is available and the type of samples you have. Table 2 outlines the most common methods to look at viability and what results you should expect.

Table 2. Methods to study cell viability

Abbreviations: LDH = lactate, PI = propidium iodide, 7-AAD = 7-aminoactinomycin D, WST1 = water-soluble tetrazolium salt, Flow Cyt = flow cytometry, IHC = immunohistochemistry, β-gal = β-galactosidase.

Ruling out apoptosis

Different approaches can be used to rule out the presence of apoptosis. These are outlined briefly below, but for more detailed information on detecting apoptosis, see our apoptosis ebook.

Caspase 3 activity

Caspase-3 is the primary executioner caspase in apoptosis, required for the mass proteolysis that leads to apoptosis. Caspases are initially synthesized as inactive pro-caspases, and they are activated by cleavage at specific sites. The method used to detect caspase-3 will depend on the available instrumentation and how the samples have been prepared (Table 3).

Table 3. Detection of caspase 3 activity

Morphological changes

The morphology of dying cells can give clues to the type of cell death occurring. This can be assessed using imaging techniques (Table 4).

Table 4. Morphological differences between apoptotic and necrotic cell death.

Secondary necrosis (associated with apoptosis) can be distinguished from primary necrosis by propidium iodide (PI) staining; homogenous PI staining indicates chromatin fragmentation indicative of apoptosis, whereas necrotic cells display staining primarily in the nucleoli.

Chromatin condensation and DNA fragmentation

During apoptosis, chromatin becomes highly condensed. When stained with DNA-binding nuclear dyes, the compacted chromatin will be brighter than the chromatin from non-apoptotic cells, and the condensed nuclei can be easily identified by fluorescence microscopy (qualitative detection) and/or flow cytometry (quantitative detection).

Condensed chromatin is then fragmented by a specific nuclease – caspase-activated DNase (CAD), generating fragments of around 200 base pairs. These can be detected by examining DNA on an agarose gel. Although this semi-quantitative method is falling out of use, it is a simple technique that provides robust answers.

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In situ direct DNA fragmentation (TUNEL) assay kit

TUNEL assay analysis. RAW 264.7 cells were left untreated (A) or treated with increasing concentrations of camptothecin (B, 2 µM; C, 10 µM) during 24 hours prior staining with In situ direct DNA fragmentation (TUNEL) assay kit (ab66108). This assay uses a deoxyuridine nucleotide labeled with FITC, which can be measured in the FL1 channel.

Apoptosis DNA fragmentation analysis protocol

Detecting necroptosis

Although many proteins are involved in the necroptotic pathway (Table 5), the most reliable method to detect necroptosis is measuring MLKL phosphorylation status and by specific inhibition of the pathway.

Table 5. Key proteins involved in necroptosis

MLKL phosphorylation

MLKL is activated by RIPK3-mediated phosphorylation. The activation state of MLKL can be determined by assessing phosphorylation status of Thr357 and Ser358. Phospho-MLKL is detected by antibody-based methods, including western blot, IHC and flow cytometry

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Anti-MLKL (phospho S358) antibody [EPR9514]

Detection of phosphorylated MLKL (pS358) by immunohistochemistry. Formalin/PFA-fixed paraffin-embedded sections (FFPE) of human skin tissue were stained with the rabbit monoclonal anti-MLKL (phospho S358) antibody [EPR9514] (ab187091) at 1:250 dilution.

Necroptosis inhibition

Targeting components of the necroptosis pathway – either by chemical inhibition or with transgenic models – can be used to tell whether cell death is dependent on them.

Table 6. Chemical inhibitors of necroptosis

Considerations for using inhibitors:

• Nec1 has some off-target activity: Nec1s is more specific²⁹.
• RIPK1 can contribute to apoptosis³⁰. Be aware that RIPK1 inhibitors may also block apoptosis under some circumstances.
• Using transgenic models is the best method for confirming the presence of necroptosis.

Detecting pyroptosis

Pyroptosis can be studied by looking at caspase activation, gasdermin D cleavage, or by inhibiting or ablating key components of the pyroptotic pathway (Table 7).

Table 7. Key proteins involved in pyroptosis

Caspase activity

Active caspases are cleaved from their inactive pre-caspase forms during pyroptosis. Caspase cleavage can be assessed by western blot and a specific caspase antibody.

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Anti-Caspase 11 antibody [EPR18628]

Detection of caspase 11 in RAW 164.7 (mouse macrophage) cell lysate with rabbit monoclonal anti-caspase 11 antibody [EPR18628] (ab180673) (1:1000 dilution). Lane 1: untreated, lane 2: cells treated with 10 mg/mL lipopolysaccharide for 8 hours. GAPDH was used as loading control.

Although active caspases are cleaved, observing caspase cleavage alone is not proof of caspase activation and other methods should also be used to confirm pyroptosis. Caspase activation can be detected directly using caspase activation assays.

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Caspase 1 Assay Kit (Fluorometric)

Activity of known concentrations of active caspase 1 (background subtracted) was detected using Caspase 1 Assay Kit (Fluorometric) (ab39412) in only two hours.

Gasdermin D

Pyroptosis involves cleavage of gasdermin D (53 kDa), resulting in a 30 kDa N-terminal fragment. Cleaved gasdermin D can be detected by western blot by the presence of a band at 30 kDa.

We recommend using our anti-gasdermin D rabbit polyclonal (ab155233), which detects the N-terminal region of gasdermin D.

Pyroptosis inhibition

Dependence on caspase 1, 11, 4 or 5 is essential to distinguish pyroptotic cell death from other forms of necroptosis and apoptosis. Determine if cell death still occurs after ablation of activity of relevant caspases, either by chemical inhibition or using transgenic models.

Caspase 1 activity can be ablated by chemical inhibition with z-YVAD-fmk (ab141388).

Detecting ferroptosis

Ferroptosis can be identified by looking at whether cell death is prevented by inhibitors, and by measuring lipid peroxides.

Table 8. Key proteins involved in ferroptosis.

Inhibiting ferroptosis

The presence of ferroptosis can be confirmed using chemical inhibitors known to prevent ferroptosis. As ferroptosis is caused by reduction of GPX4 activity, knockdown is not an effective method.

Table 9. Ferroptosis inhibitors and their modes of action

Accumulation of lipid peroxides

Ferroptosis is dependent on lipid ROS accumulation. Several methods are available to detect the presence of lipid ROS.

Table 10. Methods to detect the presence of lipid ROS

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Glutathione Peroxidase Assay Kit

Glutathione peroxidase (GPX) activity was measured in cell lysates using Glutathione Peroxidase Assay Kit (ab102530). Lysates were prepared from THP1 (human monocytic leukemia), A10 (rat thoracic aorta) and U947 (human monocytic).

References

20. Dixon, S. J. et al. NIH Public Access. 149, 1060–1072 (2013).

21. Conrad, M. & Friedmann Angeli, J. P. Glutathione peroxidase 4 (Gpx4) and ferroptosis: what’s so special about it? Mol. Cell. Oncol. 2, e995047 (2015).

28. Vanden Berghe, T. et al. Determination of apoptotic and necrotic cell death in vitro and in vivo. Methods 61, 117–129 (2013).

29. Takahashi, N. et al. Necrostatin-1 analogues: critical issues on the specificity, activity and in vivo use in experimental disease models. Cell Death Dis. 3, e437-10 (2012).

30. Kaiser, W. J. et al. RIP1 suppresses innate immune necrotic as well as apoptotic cell death during mammalian parturition. Proc. Natl. Acad. Sci. 111, 7753–7758 (2014)