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Table 2 Summary of some commonly used markers for apoptosis.

From: Investigating the evolution of apoptosis in malaria parasites: the importance of ecology

Marker of apoptosis Example of assay used Method of detection Practical considerations Relevance for malaria ecology
Activation of caspase-like molecules CaspaTag (Chemicon international, USA) A fluorescent labelled general caspase inhibitor (FAM.VAD.fmk (green) and SR.DEVD.fmk (red)) binds to active caspase within the cell. Positive cells fluoresce under fluorescent microscope - Quick and easy to use The role of caspase-like molecules is controversial in protozoan parasites, therefore it is not possible to be certain that apoptosis is being detected. However, if caspase molecules are a reliable marker they would be useful as an early marker of induction.
    -Results not as clear as with TUNEL  
    - Viability tests can be performed in conjunction  
    - Large scale experiments possible  
    The caspase inhibitor used in the caspase assay is broad spectrum and therefore may cross-react with unrelated molecules.  
Depolarisation of mitochondria outer membrane JC-1 assay kit (Molecular Probes, UK) JC-1 is a cationic carbocyanine dye that accumulates in mitochondria. Loss of mitochondrial membrane potential can be detected by the shifting of emission of fluorescence from orange (polarised mitochondrial membrane) to green (depolarised mitochondrial membrane). - Quick and easy to use The role of mitochondria in malaria apoptosis not well established. However, if markers prove to be reliable they would be useful as an early marker of induction.
    - Viability tests can be performed in conjunction  
Condensed chromatin Acridine orange (Sigma) Differentially stains SS and DS nucleic acids - enables the detection of condensed chromatin. Apoptotic cells should show an intense red staining in nucleus - Quick and easy to use Good relevance as we would expect this process to be the same for mammalian and protozoan cells.
    False positives possible and Results not as clear as with TUNEL  
    - Viability tests can be performed in conjunction  
    - Large scale experiments possible  
Translocation of phosphatidylserine to outer cell membrane Annexin V- FITC apoptosis detection kit (Sigma, UK) Positive display green annexin labelling on the cell surface, which can be detected by fluorescent microscopy - Quick and easy to use May not be relevant for malaria cells for two reasons.
    - Results not as clear as with TUNEL 1. The cell membrane of protozoan parasites is very different to that of mammalian cells.
    - Viability tests can be performed in conjunction 2. The ultimate reason for mammalian cells expressing phosphatidylserine on the outside of apoptotic bodies in order to be taken up by phagocytes, is not relevant for the mosquito midgut.
    - Large scale experiments possible  
Fragmented DNA leading to the generation of fragments with 3'OH groups In situ cell death detection kit, Flourescein (Roche) DNA of fixed and permeabilized cells labelled by the addition of flourescein dUTP at strand breaks by terminal transferase. Flourescein then detected by fluorescent microscopy (figure 3) - More laborious than using Acridine orange, CaspaTag or Annexin V detection Good relevance as we would expect this process to be the same for mammalian and protozoan cells. However as DNA fragmentation is thought to be a late process in apoptosis may only see markers at a later time point than induction of apoptosis pathways.
    - Gives clear unambiguous results.  
    - Requires cells to be dead so cannot perform viability tests in conjunction.  
    - Slides can be stored (at 5°C) for a few days allowing later analysis and therefore large scale experiments. Some necrotic cells may show positive.
Morphological Markers e.g. membrane blebbing and formation of apoptotic bodies Electron microscopy Observation of cell morphology under electron microscope to detect membrane blebing or formation of apoptotic bodies Time consuming and expensive Malaria parasite cells differ in structural aspects from mammalian cells, it is therefore not clear whether the structural changes observed in mammalian cells would be relevant for these parasites. The ultimate reasons for formation of apoptotic bodies to be taken up by macrophages also not relevant in the mosquito midgut.
    - Not a good basis for morphological changes seen in malaria apoptosis  
    - Requires cells to be dead so cannot perform viability tests in conjunction.  
    - Large scale experiments not possible but may be useful in conjunction with other assays  
Detecting cell viability
Propidium iodide (PI) Propidium iodide (Roche) Stain is taken up in cells with compromised membranes causing cells to display a red fluorescence. - Quick and easy to use Useful method for assessing viability of cells which can be used in conjunction with other assays of apoptosis.
    - Cells must be viewed quickly after application  
    - Can be used in conjunction with assays on live cells e.g. CaspaTag.  
  1. Particular reference is paid to their ecological and practical considerations when using to test evolutionary predictions. For more details on the markers of apoptosis in protozoan parasites see paper by Jiménez-Ruiz et al. ('Apoptotic markers in protozoan parasites') in this thematic.