How does calcein stain cells?
The protection of carboxylic acid groups by the hydrophobic AM (acetomethoxy) groups makes this dye readily enter viable cell. Once inside, the AM groups are removed by the intracellular esterases, trapping the calcein within the cell and giving out strong green fluorescence.
How long does calcein AM stain last?
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.
Is calcein a fluorescent?
Calcein, also known as fluorexon, fluorescein complex, is a fluorescent dye with excitation and emission wavelengths of 495/515 nm, respectively, and has the appearance of orange crystals.
Is calcein AM fixable?
Calcein, AM diffuses into cells, the ‘AM’ moiety is cleaved by cellular esterases, and then the dye molecules are observed in the cytoplasm without binding to anything. This gives a ‘whole cell’ stain. It also means that the dyes are not crosslinked with aldehyde-based fixation and thus will be lost upon fixation.
What is calcein stain?
Calcein AM is a cell-permeant dye that can be used to determine cell viability in most eukaryotic cells. In live cells the nonfluorescent calcein AM is converted to a green-fluorescent calcein after acetoxymethyl ester hydrolysis by intracellular esterases.
What is calcein labeling?
In vivo labeling of bone with fluorochromes is a widely used method for assessment of bone formation and remodeling processes. In particular, calcein is used as a marker for identification of bone growth, which is indicated by a green color.
Is calcein cytotoxic?
Calcein/AM is a fluorescent dye that localises intracellularly after esterase-dependent cellular trapping and which has shown cytotoxic activity against various established human tumour cell lines at relatively low concentrations.
Does calcein AM stain bacteria?
This stain is therefore potentially the most useful for flow cytometric analysis of viability in bac- terial populations. The inability of calcein-AM to stain many bacteria has also been reported by Kaneshiro et al.
What is calcein used for?
Is calcein AM toxic?
Calcein, AM has been used for studies of enzymatic activity, cell membrane integrity, and long-term cell tracking due to its low cellular toxicity. It is important to note that for certain cell lines and experimental conditions, relatively high concentrations of Calcein, AM can be toxic to cells.
How do you stain with calcein AM?
To stain at 1 µM, add 10 µL of the Calcein AM solution that had been further diluted 1:10 in PBS to 390 µL cells, forming a final volume of 400 µL. If different cell volumes were used, add Calcein AM appropriately. Mix by gently flicking the tubes. 10.5 Incubate cells at 37°C protected from light.
How is the dye calcein AM used in cells?
Calcein AM is a cell-permeant dye that can be used to determine cell viability in most eukaryotic cells. In live cells the nonfluorescent calcein AM is converted to a green-fluorescent calcein after acetoxymethyl ester hydrolysis by intracellular esterases.
How are fluorescein diaceate and calcein, AM used?
Calcein, AM and FDA (fluorescein diaceate) are examples of some dyes used for this application. Since these dyes are not incorporated or covalently attached to any cellular components, they may have a short retention time as some cell types may actively efflux the dye out of the cells.
How is calcein detected in a fluorescence plate reader?
Calcein AM (Figure 27.9) is a P-gp substrate, which limits its ability to enter the cells. Calcein AM is rapidly hydrolyzed inside the cells to form Calcein. Calcein is fluorescent and is readily detected using a fluorescence plate reader.
Do you need to fluorescent label cells with calcein?
The water-soluble, non-AM form of calcein (Cat. No. C481), does not require esterase cleavage to be fluorescent. I want to do a cell migration study for around 4 hours and need to fluorescently label the cells with a dye.