How to prepare samples for Annexin V Assay Background

Cell death can occur by two distinct mechanisms, necrosis or apoptosis. Necrosis occurs when cells are exposed to harsh physical or chemical stress (e.g., hypothermia, hypoxia) while apoptosis is a tightly controlled biochemical process by which cells are eliminated and where the cell is an active participant in its own termination (“cellular suicide”). 

Apoptosis is one of the main types of programmed cell death which occur in multicellular organisms and is characterized by a series of events that lead to a variety of morphological and biochemical changes, including membrane blebbing, cell shrinkage, alteration of membrane asymmetry and permeability, condensation of chromatin and nucleus, DNA fragmentation, and formation of membrane bound vesicles (apoptotic bodies) 

Reliable detection and monitoring of apoptosis is crucial for the development of treatments for apoptosis-associated diseases and for investigating apoptotic mechanisms in general. 

Translocalisation of phosphatidylserine to the outer membrane layer is an early indicator of apoptosis. Phosphatidylserine is in healthy, non-apoptotic cells predominantly located on the internal leaflet of the plasma membrane facing the cytosol (A). Early in the apoptotic process, while the cell membrane is still intact, the phosphatidylserine is translocated to the outer layer of the membrane (B). 

Annexins are group of cellular proteins that bind to phospholipids in a calcium-dependent manner, and a member of this group; Annexin V has proven to be a useful tool in detecting apoptotic cells since it preferentially binds to negatively charged phospholipids like phosphatidylserine and shows minimal binding to phosphatidylcholine and sphingomyeline. 

By conjugating a fluorescent label to Annexin V it is possible to identify and quantify apoptotic cells. Annexin V will also bind to phosphatidylserine on late apoptotic and necrotic cells but as the membrane integrity on these cells has been lost, these can be distinguished from early apoptotic cells by the use of an impermeant dye such as propidium iodide (PI) (C). 

How to prepare samples for Annexin V Assay 

Annexin V cannot be used to stain fixed cells or tissues. After staining with Annexin V and washing, cells can be fixed with 2% formaldehyde. Annexin V staining is calcium dependent, therefore 2.5 mM CaCl2 should be included in all buffers used for washing and fixation. Annexin V binds to a phospholipid in the plasma membrane, therefore staining is not compatible with alcohol-based fixation or detergent permeabilization. Follow manufacturer’s protocol for staining cells with Annexin V. Cells must be stained with Hoechst 33342, Solution 15, for detection by the instrument.

Note: Optimal cell concentration for Annexin V Assay is 2-4 x 105 cell/mL

  1. Prepare a negative control by incubating cells in the absence of an apoptosis inducing agent, but in the presence of vehicle (e.g. DMSO). 
  2. Prepare a Positive control by using an apoptosis inducing agent eg. staurosporine. 
  3. Induce apoptosis in cells using the desired method.
  4. Harvest cells after the incubation period and determine cell concentration.
  5. (Optional wash steps) Centrifuge cells at 400 for 5 minutes, discard supernatant, and resuspend pellet in 300 μL PBS. 
  6. Centrifuge cells at 400 for 5 minutes. Remove the supernatant carefully by pipetting without disturbing the cell pellet.
  7. Prepare sufficient 1X Annexin Binding Buffer. For staining and washes, approximately 1000 μL/sample.

Note: only use Annexin V buffer from this step till the end. 

  • Resuspend pellet in Annexin V binding buffer to yield a cell concentration of 2-4 x 105 cells.

Note: It is important that the cell concentrations of all samples are within the specified range in order to enable comparison between samples. 

  • Remove 96 μL of cells and add
    • 2 μL of the Annexin V-CF488A conjugate. 
    • 2 μl Hoechst 33342, Solution 15 (final concentration: 10 μg/mL)
    • Total volume 100 μL. Mix by pipetting. 
  • Incubate cells at 37° C for 15 minutes using a heating block or water bath. 

Note: This step is crucial, thus comply with the specified incubation time and temperature! Use heating block or water bath to surround the sample tube rather than placing the tube in an incubator.

  1. After 15 minutes, remove the samples from the heat block and spin down stained cells at 400 for 5 minutes at room temperature. Remove supernatant by pipetting. 
  2. Resuspend cell pellet in 300 μL Annexin V binding buffer by pipetting, centrifuge at 400 for 5 minutes and remove supernatant by pipetting.
  3. Repeat step 10 once more. 
  4. Resuspend cell pellet in 100 μL Annexin V binding buffer supplemented with 10 μg/mL PI (prepare by adding 2 μL Solution 1to 98 μL binding buffer). Sample is now ready for analysis.
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