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Cell Biology and Antibodies

Cell Viability and Cytotoxicity Assay & Cell Staining Reagents

	Fluo Cell Double Staining Kit
	Calcein, AM
	CFSE (CFDA, SE)
	BCECF, BCECF AM
	Fluorescent Phalloidins

Fluo Cell Double Staining Kit (Calcein / PI)

Fluo Cell Double Staining Kit is utilized for simultaneous fluorescence staining of viable and dead cells. This kit contains Calcein-AM and Propidium Iodide (PI) solutions, which stain viable and dead cells, respectively.

Calcein-AM, acetoxymethyl ester of calcein, is highly lipophilic and cell membrane permeable. Though Calcein-AM itself is not a fluorescent molecule, the calcein generated from Calcein-AM by esterase in a viable cell emits strong green fluorescence (excitation: 490 nm, emission: 515 nm). Therefore, Calcein-AM only stains viable cells.

Propidium iodide (PI), a nuclei staining dye, cannot pass through a viable cell membrane, thus PI stains dead cells. It reaches the nucleus by passing through disordered areas of dead cell membrane, and intercalates with the DNA double helix of the cell to emit red fluorescence (excitation: 535 nm, emission: 617 nm). Since both calcein and PI-DNA can be excited with 490 nm, simultaneous monitoring of viable and dead cells is possible with a fluorescence microscope. With 545 nm excitation, only dead cells can be observed. Since optimal staining conditions differ from cell line to cell line, we recommend that a suitable concentration of PI and Calcein-AM be individually determined.

1 Kit contains
Solution A (Calcein-AM solution, 1 mM in DMSO: 4 vials (50 µl each)
Solution B (PI solution, 1.5 mM in H₂O): 1 vial (300 µl)

References
E. S. Kaneshiro, et al., J. Microbiol. Methods, 17, 1 (1993); L. S. De Clerck, et al., J. Immunol. Methods, 172, 115 (1994); N. G.; Papadopoulus, et al., J. Immunol. Methods, 177, 101 (1994).

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Calcein, AM

Calcein-AM readily passes through the cell membrane of viable cells because of its enhanced hydrophobicity as compared to Calcein. After Calcein-AM permeates into the cytoplasm, it is hydrolyzed by esterases to Calcein, which remains inside the cell. Among other reagents, including BCECF-AM and Carboxy-fluorescein diacetate, Calcein-AM is the most suitable fluorescent probe for staining viable cells because of its low cytotoxicity. Calcein does not inhibit any cellular functions such as proliferation or chemotaxis of lymophocyte. In addition, viability assays using Calcein are reliable and correlate well with the standard 51Cr-release assay. The excitation and emission wavelengths of calcein are 490 nm and 515 nm, respectively.

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CFSE (CFDA, SE)

CFSE (5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester, also CFDA, SE) is cell membrane permeable and readily accumulates inside of viable cells where it covalently attaches to intracellular proteins. Hydrolyzed CFSE emits fluorescence and covalently-attached fluorescein molecules seldom leak from cells. CFSE-labeled cells can be monitored over several weeks in vivo. Therefore, CFSE is utilized for viable cell detection as well as for the long-term observation of cell activities by fluorescent microscopy. The excitation and emission wavelengths of CFSE-labeled cells are 500 nm and 520 nm, respectively.

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BCECF, BCECF AM

BCECF-AM is cell membrane permeable and it is easily hydrolyzed by esterases to BCECF. BCECF is not cell membrane permeable, and it accumulates inside viable cells. BCECF emits a strong green fluorescence; therefore, it is easy to visualize viable cells. BCECF-AM is also utilized as an intracellular pH indicator. The excitation and emission wavelengths of BCECF are 500 nm and 530 nm, respectively. Usually, over 80% of loaded BCECF will remain inside the cell for at least 2 hours.

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Phallotoxine

MFP488 phalloidin and MFP555 phalloidin are high-affinity probes for F-actin. Phalloidin is made from a mushroom toxin conjugated with our bright, photostable, green-fluorescent MFP488 dye and red-fluorescent MFP555 dye. Used at nanomolar concentrations, the fluorescent phalloidins are convenient probes for labeling, identifying and quantitating F-actin in formaldehyde-fixed and permeabilized tissue sections, cell cultures or cell-free experiments.

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