How does ethidium homodimer work?

Ethidium homodimer is a membrane-impermeable fluorescent dye which binds to DNA. After a cell sample has been stained with ethidium homodimer, the dead cells may be viewed and counted under a UV-light microscope. When cells die, the plasma membranes of those cells becomes disrupted.

What does ethidium homodimer stain?

Ethidium Homodimer III, also known as EthD-III, is a red fluorescent dead cell stain for bacteria and mammalian cells. It is a cell membrane-impermeant nucleic acid dye that stains only dead cells with damaged cell membranes.

Is ethidium homodimer toxic to cells?

Ethidium Homodimer I dye (EthD-I) is highly positively charged, it cannot cross cell membranes to stain living cells.

How does calcein work?

Calcein AM is a non-fluorescent cell-permeable derivate of calcein that is widely used in cell viability measurement. Once inside cell, AM groups are hydrolyzed by intracellular esterases. The fluorescent calcein molecule is restored, which is trapped in the cell and emits strong green fluorescence.

Is calcein AM fixable?

Note: In general, DNA-binding dyes and calcein AM are not compatible with fixation, as these dyes are not covalently bound to components of the cell and will thus slowly diffuse out of cells after fixation, gradually staining all cells as dead.

How long does calcein AM last?

We use Calcein AM to allow us to take images of neuronal processes for analysis of their length and branching. Over the short term (12-24 hours) Calcein AM does not seem to kill neurons. Some minimal neuronal death is seen by 48 hrs of culture with Calcein AM and this is increased by 72 hrs culture.

How does ethidium bromide stain to DNA?

The most commonly used stain for detecting DNA/RNA is ethidium bromide. Ethidium bromide is a DNA interchelator, inserting itself into the spaces between the base pairs of the double helix. Ethidium bromide possesses UV absorbance maxima at 300 and 360 nm.

What happens if you touch ethidium bromide?

Health and Safety EtBr is a potent mutagen (can cause genetic damage), and moderately toxic after an acute exposure. EtBr can be absorbed through skin, so it is important to avoid any direct contact with the chemical. The powder form is considered an irritant to the upper respiratory tract, eyes, and skin.

Is DAPI a counterstain?

DAPI is a popular nuclear counterstain for use in multicolor fluorescent techniques. Its blue fluorescence stands out in vivid contrast to green, yellow, or red fluorescent probes of other structures. When used according to our protocols, DAPI stains nuclei specifically, with little or no cytoplasmic labeling.

Does DAPI stain glia?

Cultured cells usually change their outward shape dramatically after being isolated so that it is frequently impossible to tell neurons and glia apart. Thus, double labeled cells can be identified as neurons, cells that carry only DAPI staining are non-neuronal.

What can ethidium homodimer III be used for?

Ethidium Homodimer III is membrane impermeant and useful for selectively staining dead cells with damaged plasma membranes. It also is a component of several combination viability assay kits for detecting both live and dead cells in the same population, such as our Viability/Cytotoxicity Assay Kit for Animal Cells (Cat.

How are calcein AM and ethidium homodimer stained?

Damaged or dead cells are alternatively stained by ethidium homodimer, a membrane impermeable molecule and fluorescent red when the dye binds to nucleic acids. Calcein AM and ethidium homodimer emit distinct fluorescence signatures at 515 nm and 635 nm respectively when excited at 495 nm [107,108].

What is the fluorescence signature of ethidium homodimer?

Calcein AM and ethidium homodimer emit distinct fluorescence signatures at 515 nm and 635 nm respectively when excited at 495 nm [107,108]. Furthermore, the power of visual inspection was also proven by McMurtrey et al. [109], wherein there was shown to be significant control of aligned nanofibers over the direction of neurite outgrowth.

How are ethidium and propidium used to detect DNA?

In recent years, the DNA-intercalating dyes, such as ethidium monoazide (EMA) and propidium monoazide (PMA), have been used to overcome the problem of detecting DNA from dead cells. The principle of EMA and PMA is based on the integrity of cell membranes.