On 18 Nov 2002 02:41:44 -0000, tflo at systemsbiology.org
<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.
If you can't detach the cells (why not?), but can set up your
experiments in 96-well plates, you might want to try using an
ELISA-type assay. For example, if you can conjugate AnnexinV to alkaline
phosphatase (if you can't do it directly, you can use biotin-AnnexinV
and avidin-AP, or even get FITC-AnnexinV and then use an anti-FITC
antibody,etc.), you can quantitate AnnexinV binding using a standard
spectrophotometric assay for AP activity. It won't give the number of
dead/dying cells, but it may allow you to get a measure of cell death.
You may also be able to adapt TUNEL to this kind of assay. Conversely,
since many cell-surface proteins are lost when cells die, you can
quantitate live cells with a primary Ab against some macrophage marker
followed by an AP-linked secondary. This may work particularly well if
the dead macrophages become detached, since then you don't even have to
worry about whether they still express the marker in question - they'll
get washed out of the wells anyway.
I hope this is helpful,
Ken Frauwirth
--
Ken Frauwirth (MiSTie #33025) kfrauwir at mail.med.upenn.edu
Abramson Cancer Research Institute
University of Pennsylvania
http://mail.med.upenn.edu/~kfrauwir
"Science in those days worked in broad strokes. They got right to the
point. Nowadays it's just molecule, molecule, molecule."
The Tick
