Cell viability or death assay

Dr Engelbert Buxbaum engelbert_buxbaum at web.de
Tue Nov 19 04:50:03 EST 2002


tflo at systemsbiology.org wrote:

> Hi,
> I have a problem that I hope you can help me with. I am doing in vitro
> infections of primary macrophages with various live intracellular bacteria, and
> I have great problems in finding a method to evaluate bacteria-induced cell
> death of the macrophages / cell viability. To summarise:

Viability assays all have their limitations, but 4 types are generally
used:

1) MTT: Forms a red formazan dye in the presence of healthy cells,
specifically, healthy mitochondria. This dye is dissolved in acidic
propanol and measured in a photometer. The assay can be performed in
96-well plates, and is probably the most commonly used. Parameter:
mitochondrial metabolism

2) Fluorescein diacetate: Is membrane permeable and non-fluorescent. In
the cytosol it is converted into fluorescein by cellular esterases,
which is not membrane permeable and accumulates inside the cells. The
fluorescence is visible under a fluorescence microscope, or in a FACS.
Parameter: membrane integrity

3) Trypan blue exclusion: The dye is dissolved in PBS and mixed with the
cells, usually on a haemocytometer. Healthy cells with intact membranes
will exclude trypan blue and appear white against a blue background.
Dead cells will also appear blue. Parameter: Membrane integrity

4) Incorporation of tritiated desoxy-nucleotides: As the cells divide,
they make new DNA. Thus radioactive desoxy-nucleotidess are incorporated
into an acid-insoluble form and can be counted. Parameter: cell division
(so not suitable for your problem).



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