5(6)-CFDA, SE (CFSE)

Calcein AM (MW: 994.87) is a cell membrane-permeable, non-fluorescent dye that is hydrolyzed by esterases to the cell membrane-impermeable green-fluorescent Calcein. Since esterases are only active in viable cells, the green fluorescence is directly proportional to the number of live cells in culture.
General Staining Procedure:1. Prepare 1 mM Calcein-AM solution with DMSO and dilute to prepare 1-50 µM Calcein-AM solution with PBS.2. Add Calcein-AM solution with 1/10 of the volume of cell culture medium to the cell culture.3. Incubate the cell at 37ºC for 15-30 minutes.4. Wash cells twice with PBS or an appropriate buffer.5. Observe the cells using a fluorescence microscope with 490 nm excitation and 515 nm emission filters.

Related Links and Documents

• Calcein AM
• Live/Dead Cell Staining Kit II
• Kurs Zellviabilitäts-, Proliferations- und Toxizitätstests (German)

No, it does not stain viable cells because a water-soluble tetrazolium salt (WST-8) is used for the solution. The electron mediator, 1-Methoxy PMS, receives electrons from viable cells and transfers the electrons to WST-8 in the culture medium. Since its formazan dye is also highly water soluble, CCVK-I cannot be utilized for cell staining.

Related Links and Documents

• Colorimetric Cell Viability Kit I (WST-8)

This kit has been used successfully on gram positive and gram negative bacteria, but we don’t have any knowledge on whether it will work with cyanobacteria.

Related Links and Documents

• Bacteria Live/Dead Staining Kit

Our Live/Dead Cell Staining Kit is for use in unfixed living cells, so it cannot be used in paraffin sections or cryosections of tissue.
There are some references reporting the use of calcein-AM and vital dyes in dissected tissues or organ cultures in vitro.  The main concern would be whether the dyes can penetrate the areas of tissue that you want to examine. Also, calcein is not fixable, so you would need to observe the fluorescence in the tissue without additional processing. A longer incubation time might be required in tissue specimens compared to cells to allow the dyes to penetrate and for conversion calcein-AM into calcein. Also, because ethidium homodimer III stains cell with compromised membrane integrity, it will likely stain cells along cut edges or on the surface of a tissue slice.
A fixable alternative to calcein-AM is CFSE, which is non-fluorescent until it is hydrolyzed by esterases in the cytoplasm of living cells to generate fluorescein-SE, which covalently attaches to cellular proteins. We offer CFSE (Cell Proliferation Kit I, PK-CA707-30050), which you could pair with a vital dye like propidium iodide or ethdium homodimer III to stain dead cells. For the vital dyes, it is important to wash away the free dye thoroughly before fixing to avoid background staining.

Related Links and Documents

• Live/Dead Cell Staininng Kit II
• Cell Proliferation Kit I (CFSE)

Cellular senescence limits the lifespan of mammalian cells, prevents unlimited cell proliferation and is thought to be a tumor suppressive mechanism. In the cell culture dish, senescence represents a state in which the cells remain viable, but loose the ability to divide. Furthermore, the cells display an increase in size, senescence-associated expression of beta-galactosidase (SA-beta-Gal) activity, and altered patterns of gene expression.

• The PromoKine Senescence Detection Kit I is designed to histochemically detect SA-beta-Gal activity in fixed cultured cells and tissue sections. Senescent cells develop a blue color precipitate that can be observed under the microscope.
• The Senescence Detection Kit II is designed to fluorescently detect SA-beta-Gal activity in cultured cells by FACS (FL1 channel)

SA-beta-Gal is present only in senescent cells and is not found in presenescent, quiescent or immortal cells.

Related Links and Documents

• Senescence Detection Kit I
• Senescence Detection Kit II

For adherent cells, at least 1,000 cells are necessary per well (100 µl medium) when using a standard 96-well plate. For leukocytes and other suspension cells, at least 2,500 cells/well (100 µl medium) are necessary because of lower sensitivity. The recommended maximum number of cells per well for the 96-well plate is 25,000. For 384 well plates, just divide these cell numbers by 4 (250 cells per well for adherent,  625 cells per well for suspension cells). If 24-well or 6-well plates are used for the assay, please calculate the number of cells per well accordingly, and adjust the volume of the WST-8 solution in each well to 10% of the total volume. For more exotic culture systems, like 3-D models or sphereoids, you  should start with 10% WST-8 reagent per vol. of media and culture until you get a good reading. Some  optimization of the protocol may then be necessary because the cells‘ metabolisms behave differently in 3-D. 

Related Links and Documents

• Colorimetric Cell Viability Kit I (WST-8)
• Kurs Zellviabilitäts-, Proliferations- und Toxizitätstests (German)
• Kurs Zytotoxizität und Mutagenität (German)