For general safety in laboratory work, consider the general document RWS 004 on environmental safety and work security. Calcein AM and Ethidium homodimer are irritating to eyes, respiratory system and skin. Wear suitable protective clothing.
2.Rationale
The viability assay provides a two-color fluorescence cell viability test that is based on the simultaneous determination of live and dead cells with two probes that measure two recognized parameters of cell viability – intracellular esterase activity and plasma membrane integrity. The assay is applicable to most eukaryotic cell types, including adherent cells and certain tissues, but not to bacteria or yeast.
Live cells are distinguished by the presence of ubiquitous intracellular esterase activity, determined by the enzymatic conversion of the virtually non-fluorescent cell-permeant calcein AM to the intensely fluorescent calcein. Calcein is well retained within live cells, producing an intense uniform green fluorescence in live cells (ex/em ~495 nm/~515 nm). Ethidium homodimer-1 enters cells with damaged membranes and undergoes a 40-fold enhancement of fluorescence upon binding to nucleic acids, thereby producing a bright red fluorescence in dead cells (ex/em ~495nm/635 nm).
3.Application
To be performed by persons trained in cell biology and fluorescence microscopy after qualification by an instructor
4.Applicable documents
No special
5.Definition
No special
Stock solution: 1 mg/ml in 20% DMSO in H2O; store in small aliquots at -80℃.
·Fluorescence microscope
Calcein and EthD-1 can be viewed simultaneously with a conventional fluorescein longpass filter. The fluorescence from these dyes may also be observed separately; calcein can be viewed with a standard fluorescein bandpass filter and EthD-1 can be viewed with filters for propidium iodide or Texas Red®. Consider the general document on fluorescence microscopy for specifications of the microscope and the optical filters to be used.
7.Procedure
Adherent cells may be cultured on sterile glass coverslips inside 35 mm disposable petri dishes. Wash the cells prior to the assay to remove or dilute serum esterase activity generally present in serum-supplemented growth media (serum esterases could cause some increase in extracellular fluorescence by hydrolyzing calcein AM).
·Prepare working solution of 1 µl/ml of each stock solution in sterile PBS or DMEM
·Incubate cells in working solution at 37℃ for 20 min
·View the labeled cells under the fluorescence microscope
8.Documentation, Archiving
Pictures are saved as tif-files in the project files
9.Special Requirements, Quality Assessment
Aqueous solutions of calcein AM are susceptible to hydrolysis. Working solutions should therefore be used within one day.
10.References
LIVE/DEADÒ Viability/Cytotoxicity Kit (L-3224) for animal cells, Product Information, Molecular Probes, 2001
Papadopoulos NG et al. An improved fluorescence assay for the determination of lymphocyte-mediated cytotoxicity using flow cytometry. J Immunol Methods 177 (1-2): 101-11, 1994.
