Annexin Vivo on ASK


Overview

Programmed cell death, or apoptosis, is an important component of various cellular processes. These include embryonic development, hormone-dependent atrophy, normal mature cell turnover, proper development and functioning of the immune system. Apoptosis can also occur as a defense mechanism, such as in immune responses or when cells are damaged by disease or chemical agents. However, many human diseases and conditions can be linked to an increase or decrease in the normal levels of apoptosis. For example, neurodegenerative diseases, ischemic damage, autoimmune disorders and many types of cancer have been attributed to inappropriate levels of apoptosis. Apoptotic cells can be recognized by defined morphological changes such as cellular shrinking, chromatin condensation, and eventually cell fragmentation into small apoptotic bodies.

One of the early events in apoptosis is the translocation of phosphatidylserine (PS) from inside the cell to the outer membrane. This change provides a signal to macrophages favoring phagocytosis over the inflammatory response. The PS translocation in apoptosis can be detected by using labeled annexin V. Annexin V is a 36 kDa human protein that binds specifically, in a calcium-dependent manner, with nanomolar affinity to externalized PS.

Annexin-Vivo™ 750 was developed by conjugating annexin V with a NIR fluorophore. This agent has been developed to enable in vivo visualization and quantification of the membrane-bound phospholipid, PS, exposed in the outer leaflet of the cell membrane lipid bilayer during the early stages of apoptosis. It can provide an effective in vivo imaging tool for the rapid detection of therapeutic efficacy in a variety of pathological conditions (including cancer, stroke, atherosclerosis, myocardial ischemia, and liver toxicity), facilitating the discovery and characterization of novel therapeutics.


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Figure 1: Diagram of annexin V function in apoptosis. During apoptosis, phosphatidylserine (PS) is translocated from the inside of the cell to the outer membrane. Annexin V can then bind PS in a calcium-dependent manner.



Products and catalog numbers

ProductCatalog NumberEx/Em wavelength
(nm)		Molecular weight (g/mol)Validated ExperimentsApplicationsStorage and Stability
Annexin-Vivo™ 750NEV11053755/77235,000In vivo/Ex vivo
Flow cytometry
In vitro microscopy		Oncology
Cardiovascular
Transplant Rejection
Pancreatitis		Technical Data Sheet


Using Annexin-Vivo 750 in vivo/ex vivo

The generally recommended procedure for in vivo imaging with Annexin-Vivo 750 is administration via intravenous injection and imaging 2 hours post injection. Annexin-Vivo 750 will clear from tissues after ~3 days after which repeat injection and imaging may be performed for longitudinal studies. However, it is recommended that a pre-injection baseline image be taken prior to reinjection and imaging.


Route of InjectionMouse Dose (25 g)Rat Dose (250 g)Blood t 1/2Tissue t 1/2Optimal imaging timeOptimal Re-injection Time (complete clearance)Route of Metabolism/ background tissueFMT& IVIS settings
IV100 uL300-1000 ug5 min142 h3 dkidneys (high), liverFMT 750/770
IVIS 745/800

In Vivo Imaging

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Figure 2: CY-treated and untreated HT-29 tumor xenograft mice were injected with Annexin-Vivo™ 750 and imaged two hours later by FMT 2500TM. Figure shows representative images of treated and untreated HT-29 mice selected to reflect the mean group tumor fluorescence intensities. A) The 2D planar fluorescence images are shown. B) The 3D FMT images show all signal in and around the tumors. C) Representative FMT images in HT-29 tumor xenografts are shown from CY-treated mice (upper panels) and control (lower panels), showing only tumor-associated signal. D) Quantification of absolute pmols per tumor in control (n = 26) and CY-treated (n = 28) tumors. The tumor volume data confirms that there was no treatment effect on tumor volumes (as assessed by dimensional caliper measures).


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Figure 3: To assess the optimal time-point for imaging Annexin-Vivo™ 750 in CY-treated tumors, control (A, n=12) and treated (B, n=13) mice bearing HT-29 tumor xenografts were injected with the agent and imaged 2, 4, 24, and 48 hours later.


Ex Vivo Imaging

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Figure 4: Ex vivo TUNEL staining of tumors from CY-treated and untreated HT-29 tumor xenografts. Tumor tissues were removed from HT-29 tumor xenograft mice, frozen, and sectioned for TUNEL staining. A) 400X microscopy images of TUNEL-stained tissues. B) Cell count results from multiple quadrants of sectioned tissue, represented as staining of total cells.



Using Annexin-Vivo 750 in vitro

Annexin-Vivo 750 has been validated for use in vitro with tumor cells (Jurkat A3).

Flow cytometry and in vitro microscopy

We have validated Annexin-Vivo 750 for use with fluorescence microscopes and flow cytometers. Here is a brief protocol with a recommended concentration of agent to use:

  1. Resuspend cells in complete medium. Treat with 200 ng/ml anti-FAS antibody for 3-4 h prior to incubation with agent. 
  2. Incubate cells with 1 µM Annexin-Vivo™ 750 for 10 min at 37 °C.
  3. Wash 1x with PBS. 
  4. Flow cytometry filter settings: Add Propidium iodide 1 min prior to acquisition 575/26; 780/60
    Fluorescence micrsocopy filter: Cy5.5 or Cy7*

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Figure 5: A. Annexin-Vivo™ 750 was incubated in vitro with treated and untreated Jurkat cells and labeling was compared to TUNEL staining of cells (B). C. Cell counts of TUNEL positive cells by microscopy. D. Jurkat cells induced to apoptosis by anti-CD95 (Anti-FAS) were incubated with Annexin-Vivo™ 750 and assess by flow cytometry. E. Cells in early apoptosis (Annexin-Vivo™ 750 positive only) versus late apoptosis. Annexin-Vivo™ 750 and propidium iodide double-positive were counted and represented as a percentage of total cells.



Citations

Please visit our Citations Library for references using Annexin-Vivo 750 on the IVIS or on the FMT.