Quick Answer: How Does Annexin V Work?

How does propidium iodide staining work?

Propidium iodide (PI) is a membrane impermeant dye that is generally excluded from viable cells.

It binds to double stranded DNA by intercalating between base pairs.

PI is excited at 488 nm and, with a relatively large Stokes shift, emits at a maximum wavelength of 617 nm..

What does Tunel stand for?

terminal deoxynucleotidyl transferaseTUNEL stands for terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. The two main TUNEL reagents are terminal deoxynucleotidyl transferase, or TdT, and deoxyuridine triphosphate, or dUTP, which may be fluorescently labeled for ease of detection.

How is cell death calculated?

Cell death can be measured by staining a sample of cells with trypan blue, as we describe in Protocol: Measuring Cell Death by Trypan Blue Uptake and Light Microscopy (Crowley et al. 2015b).

Can propidium iodide be fixed?

Propidium Iodide is a charged substance; i.e. the positively charged propidium ion does not enter living cells. If the aim is to stain all cells, then they must be fixed in ethanol before staining.

Is annexin V an antibody?

Annexin V Antibody recognizes endogenous levels of total annexin V protein. This antibody is not predicted to cross-react with other annexin family members.

How does a Tunel assay work?

Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay has been designed to detect apoptotic cells that undergo extensive DNA degradation during the late stages of apoptosis. The method is based on the ability of TdT to label blunt ends of double-stranded DNA breaks independent of a template.

What is the apoptosis pathway?

Apoptosis can be initiated through one of two pathways. In the intrinsic pathway the cell kills itself because it senses cell stress, while in the extrinsic pathway the cell kills itself because of signals from other cells. Weak external signals may also activate the intrinsic pathway of apoptosis.

What is Annexin V assay?

In molecular biology, an annexin A5 affinity assay is a test to quantify the number of cells undergoing apoptosis. The assay uses the protein annexin A5 to tag apoptotic and dead cells, and the numbers are then counted using either flow cytometry or a fluorescence microscope.

What does Tunel measure?

TUNEL is a method for detecting apoptotic DNA fragmentation, widely used to identify and quantify apoptotic cells, or to detect excessive DNA breakage in individual cells. … It may also label cells having DNA damaged by other means than in the course of apoptosis.

What is the definition of apoptosis?

A type of cell death in which a series of molecular steps in a cell lead to its death. This is one method the body uses to get rid of unneeded or abnormal cells. The process of apoptosis may be blocked in cancer cells. Also called programmed cell death.

How do you make an Annexin V binding buffer?

The Annexin V Binding Buffer is a 10X concentrate composed of a 0.2 µm sterile filtered 0.1M Hepes (pH 7.4), 1.4M NaCl, and 25 mM CaCl2 solution. Prior to staining cells, an appropriate quantity of a 1X working solution should be made by diluting the 10X concentrate 1:10 with distilled water.

Does DAPI stain live or dead cells?

It is essentially excluded from viable cells, but can penetrate cell membranes of dead or dying cells. However, at high concentrations or prolonged incubation times DAPI may still enter viable cells.

What does DAPI stain for?

DAPI (4′,6-diamidino-2-phenylindole) is a blue-fluorescent DNA stain that exhibits ~20-fold enhancement of fluorescence upon binding to AT regions of dsDNA. … DAPI is generally used to stain fixed cells since the dye is cell impermeant, although the stain will enter live cells when used at higher concentrations.

What is an apoptosis assay?

An apoptosis assay detects and quantifies the cellular events associated with programmed cell death, including caspase activation, cell surface exposure of phosphatidylserine (PS) and DNA fragmentation. … The Caspase-Glo® 3/7, 8 and 9 Assays are the most sensitive caspase assays available.

How do you detect apoptosis?

There are several spectroscopic techniques available to study apoptosis, including annexin V staining, the TUNEL assay, caspase detection, and measurement of mitochondrial membrane potential. Labeled annexin V can be applied in both flow cytometry and light microscopy to identify mid- to late-stage apoptotic cells.

Can you fix annexin V?

In my experience, it is possible to fix annexin V stained cells and see great results on the flow. Stain cells with annexin V (in calcium-rich binding buffer). … As soon as you reduce calcium, the AxV simply falls off. Washing cells can induce some changes in cells as, if they are undergoing apoptosis.