Webinar: Accelerate the Detection and Imaging of Apoptotic Cells with Apotracker™ Green (Apo-15)

In the human body, billions of cells die every day; this cell death is termed apoptosis. Clearance of these cells is crucial for the maintenance of homeostasis, and its dysregulation can lead to inflammatory diseases and cancer. However, many optical probes for the detection of apoptotic cells are incompatible with physiological conditions and in vivo assays. In this webinar, Dr. Nicole Barth and Prof. Marc Vendrell of the University of Edinburgh will describe how Apotracker™ Green (also known as Apo-15 peptide) was optimized to detect and image apoptosis in real time. Apotracker™ Green is a highly stable fluorogenic peptide that selectively stains apoptotic cells in a calcium-independent manner and under wash-free conditions. They will discuss their approach to identify its molecular target and this marker for fluorescence-based assays, including in vitro assays and in vivo mouse models of lung inflammation and breast cancer.

What you will learn:

• How Apotracker™ Green was designed, optimized, and validated
• Advantages and limitations compared with other reagents for detection and imaging of apoptotic cells
• How to choose the best reagents to detect apoptosis

Additional Q&A With Drs. Barth and Vendrell

Due to great interest in this talk, many submitted questions weren’t answered during the webinar. Drs. Barth and Vendrell graciously answered these questions, below.

1. Do you need to use fresh tissue or can you used fixed tissue?
   
   Fresh tissue should be used preferably, as the fixation may interfere with the composition of the tissue.

    

2. Do you Acetone fix cryosections?
   
   For the tissue images, we fixed the tumors for 2h with 4% PFA, followed by a sucrose gradient and embedding of the tissue in OCT. If you require more detailed protocols, please don't hesitate to contact us.

    

3. Is Apo-15 highly specific for apoptosis or would another types of cellular death appear as positive?
   
   As with Annexin V and other probes that detect the exposure of phosphatidylserine, it may also detect other types of cell death if these are characterized by membrane alterations such as the exposure of phosphatidylserine. There is no probe that can solely determine that a cell death is apoptotic; a combination of reagents is normally needed to confirm this. However, the use of the detection of phosphatidylserine has been widely accepted and is the gold standard for the detection of apoptosis.

    

4. Hello congratulations for the talks! Could you comment about how to select a peptide for staining with Trp_BODIPY?
   
   The selection of peptides to be labelled with Trp-BODIPY can be done in many different ways: 1) reports in the literature of known peptides for your targets, 2) de novo construction of peptides and evaluation of their binding to the targets.

    

5. How many washes can Apotracker tolerate?
   
   The most elaborate protocols we used were for the caspase staining of the OCT-embedded sections in which we washed the tissue about 15 times. However, for flow cytometry the most washing steps that we have carried out were around eight. For both protocols, we were able to detect a clear Apotracker/Apo-15 positive population. The retention of binding may also depend on the concentration of the probe or the amount of cells.

    

6. What route did you administer Apo-15 to mice? iv or ip any different efficacy?
   
   Apo-15 was administered either i.t. or i.v., we have never carried out a comparison in staining and signal between different routes of administration.

    

7. How would cell-mediated killing affect the staining? Such as degranulation by CAR-T cells. Would the dye still be visible after cell-mediated death?
   
   It depends on the type of cell death. T cell-mediated immune responses and cell-mediated death often include the activation of Caspase-8/Caspase-3 through granzyme B entering the cancer cells. In this case, caspase-3 activation will lead to the exposure of phosphatidylserine on the cell membrane and you will be able to stain the apoptotic cancerous cells with Apo-15/Apotracker. Taken together, Apo-15 should detect cell death upon CMD.

    

8. Comment: I suggest the presenters read about C2Am, an imaging agent based on the C2A domain of SYTI, which is a small (16 kDa) protein, stable in serum, potent against PS (20 nM) and has been used in vivo for PET imaging.
   
   Thanks for the comment, Andre. We know C2Am well as well as other proteins (annexins, C2 lactadherin, etc) that have been used for PET imaging of apoptosis. Apo-15 is a complementary tool to those, enabling in vitro and in vivo optical imaging experiments.

    

9. Considering its small size, does Apo-15 penetrate and stain properly 3D forms?
   
   We have never tried it but from our educated guess we would reckon that Apo-15 can cross several layers of 3D structures.

    

10. Lovely talk! There's some evidence that T cells will display phosphatidylserine reversibly upon activation by antigen. Does Apo-15 also stain these or does it only pick up dying cells which are genuinely dying?
    
    This is a very interesting question. Phosphatidylserine has been detected on a wide range of activated cells including T cells, B cells and macrophages. However, the pathway of phosphatidylserine exposure is different from the one you find in apoptotic cells. Phosphatidylserine exposure in viable cells is widely dependent on a Ca2+-dependent scramblase TMEM16F what is reversible, whereas in apoptotic cells it is mediated through the Caspase-3 dependent inactivation of flippases AT11A/C and activation of Xkr8/9 scramblase. This leads to the levels of exposure of phosphatidylserine on activated cells to be generally lower than on dead cells in order to avoid their clearance by phagocytes. I would think that the levels on viable cells won't be near as high as apoptotic cells and therefore, Apo-15 will be able to discriminate between those.

     

11. Are apoptotic cells dead cells or a stage between live and dead cells?
    
    Apoptotic cells are dead cells. Apoptosis belongs to one of the three main cell death categories as determined by the Nomenclature Committee on Cell Death. The first morphological alterations have been first described by Kerr et al. in 1972. Apoptosis is a regulated cell death; it relies on a dedicated molecular machinery and is generally anti-inflammatory. If interested, a good review to read is Galluzzi et al., 2018, Cell Death & Differentiation, as well as most of the reviews by D.R Green, e.g. Cell Death Signaling, CSH Perspective in Biology, 2015.

     

12. I looked over your paper using the MMTV-PyMT mouse model with this reagent.  My question is, do you think that this reagent could be injected into mice and then measured non-invasively on a machine such as an IVIS or Lago X scanner?
    
    We have used it in vivo for intravital imaging but not in whole-body imaging. The emission wavelength of Apo-15 is around 520 nm so it may be short for use in IVIS or Lago X.

     

13. I was very interested in this product and I was wondering if you know if this works in live zebrafish larvae?
    
    It may depend on the experiment, but we have done some positive preliminary experiments in zebrafish larvae.