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Cat. No.: CPCVCA-100 (for 100T)

Cat. No.: CPCVCA-500 (for 500T)

Cat. No.: CPCVCA-2500 (for 2500T)

Description

Calcein/PI Cell Viability/Cytotoxicity Assay Kit (or Calcein/PI Live/Dead Viability/Cytotoxicity Assay Kit) is a highly convenient reagent kit for detecting the viability of animal cells based on the dual fluorescence staining method of Calcein-AM (calcein acetoxymethyl ester) and PI (propidium iodide).

This assay kit is easy to use, and both staining and detection take only about 30 minutes. After 30 minutes of staining with this assay kit, subsequent fluorescence microscopy imaging, flow cytometric analysis, or quantitative fluorescence detection and analysis using a fluorescence microplate reader can be performed.

The principle of this assay kit lies in the ability of the two probes to respectively detect intracellular esterase activity and cell membrane integrity, reflecting cell viability and cytotoxicity. Specifically, Calcein AM stains live cells, exhibiting green fluorescence, while Propidium Iodide (PI) stains dead cells, exhibiting red fluorescence.

Calcein AM (Calcein Acetoxymethyl Ester is a cell staining reagent that can fluoresce when binding to live cells. Derived from Calcein, Calcein AM has an added acetoxymethyl (AM) group, enhancing its hydrophobicity, thus facilitating easy penetration through the cell membrane. Calcein AM itself is non-fluorescent. However, once inside the cell, it is hydrolyzed by endogenous esterases in live cells to form the polar molecule calcein, which carries a strong negative charge and cannot pass through the cell membrane, thereby becoming trapped inside the cell. Calcein emits strong green fluorescence. Since dead cells lack esterases or have low esterase activity, Calcein AM entering dead cells cannot generate calcein, or only produces minimal amounts, resulting in weak or no staining. Therefore, only live cells are stained with intense green fluorescence, while dead cells remain unstained or exhibit very weak staining.

Propidium Iodide (PI), a red fluorescent nucleic acid stain, cannot penetrate the intact membranes of live cells but can stain the nuclei of dead cells where the membrane integrity has been compromised. Therefore, when Calcein AM and PI are used together, live cells are stained green, and dead cells are stained red, enabling dual fluorescence staining for the detection of cell viability and cytotoxicity.

The maximum excitation and emission wavelengths for the hydrolysis product of Calcein, calcein, are 494nm and 517nm, respectively. For the PI-DNA complex, the maximum excitation and emission wavelengths are 535nm and 617nm, respectively.

This assay kit can be used for the detection of most mammalian cells. There are reports indicating that Calcein AM can also be used for certain plant cells such as root border-like cells in Arabidopsis, some yeasts like Pichia anomala and Saccharomyces cerevisiae, and some parasites like Leishmania. Due to the unique composition of the cell membranes in some parasites, Calcein AM may not enter the corresponding live cells, but it can enter early apoptotic parasite cells, thus, when combined with PI, it can be used to detect early apoptosis in parasite cells. Since fungi and bacteria have cell walls that hinder the entry of Calcein, Calcein AM is not suitable for use with fungi and bacteria. In comparison to similar staining solutions such as Trypan Blue, this assay kit is quicker and more sensitive.

The Calcein AM and PI provided in this assay kit are in 1000X solutions, making them very convenient to use. Both solutions are optimized for most cells, but for optimal results, adjustments to the dilution of Calcein AM or PI can be made between 500-2000 fold depending on cell type and experimental conditions. Additionally, the assay kit provides a detection buffer that can maintain the normal state of cells for a period of time and provide some nutrition to the cells, which is more effective than PBS or HBSS.

For a 96-well plate, prepare the relevant assay reagents according to the recommended dilutions, and use 100μl per well. With this setup, the small, medium, and large packaging of this assay kit can perform 100, 500, and 2500 tests respectively.

Components

• Calcein AM (1000X) 
• PI (1000X) 
• Detection Buffer

Storage

Store at -20°C, valid for one year. Calcein AM (1000X) and PI (1000X) should be protected from light.

Notes

• Fluorescent dyes are prone to photobleaching. Please avoid light exposure as much as possible to slow down photobleaching.
• Calcein AM (1000X) is susceptible to decomposition in humid environments. It is recommended to aliquot appropriately and store sealed at -20°C upon first use.
• Due to the instability of Calcein AM in aqueous solutions, the Calcein AM detection working solution should be prepared immediately before use and cannot be frozen after preparation.
• Serum and phenol red in the culture medium may have some impact on the staining of Calcein AM, resulting in enhanced fluorescence background. It is recommended to wash the cells appropriately before adding the Calcein AM detection working solution.
• This product is for scientific research use by professionals only and is not intended for clinical diagnosis or treatment, food, or pharmaceutical purposes. It should not be stored in residential areas.
• For your safety and health, please wear laboratory attire and disposable gloves during operation.

Only for research and not intended for treatment of humans or animals

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