Calcein AM refers to a cell-permeant dye that has been widely used for the determination of cell viability. In the living cells, non-fluorescent calcein AM is converted to green fluorescent calcein following hydrolysis of acetoxymethyl ester by intracellular esterases. In this case, the calcein release method can be served as a fluorescent equivalent of the 51Cr-release assay. What’s more, preloading cells with calcein does not affect cell proliferation and other functions. Here, Creative Biolabs has developed the novel calcein release assay protocol to promote your research. Please note that our protocols are only for your reference.
Calcein Release Assay Protocol
Fig.1 Flow chart of the calcein release assay protocol.
As a famous expert with a high reputation in the complement system market, Creative Biolabs describes a variety of protocols for our clients. Along with our complement-related products and services, we can work with you to create a timeline and plan for your operation. For customers' special project needs, we offer a variety of solutions to fit your specific research needs.
Published Data
Fig 2. (A) Washing of Calcein AM-labeled plasma EVs does not significantly change the fluorescence intensity. (B) Significant loss of EVs after each washing of plasma EVs.1
Extracellular vesicles (EVs) are secreted by cells, composed of a phospholipid bilayer, usually contain bioactive molecules, and act in cell-to-cell communication. Previous use of flow cytometry with fluorescent staining to detect extracellular vesicles has some limitations, such as the possibility of false detection of cell fragments or the need to know the cell source in advance. Here, Calcein AM is demonstrated to be suitable for detecting and quantifying EVs from various sources by flow cytometry. Before entering EVs, Calcein AM is non-fluorescent. After entering EVs, EVs are activated to fluoresce and cannot penetrate EVs. The Calcein AM method can distinguish between intact EVs and EV fragments. The method has a wide range of applicability, and Calcein AM has been observed to detect EVs from at least two species, multiple cell types, cultured cells in different states, and human circulation in different health states.
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Reference
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Gray, Warren D., Adam J. Mitchell, and Charles D. Searles. "An accurate, precise method for general labeling of extracellular vesicles." MethodsX 2 (2015): 360-367. Distributed under Open Access license CC BY 4.0, without modification.
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