If what you want to measure is bacteria-induced apoptosis then why not take
macrophage samples (wells), detach them, and do an IP/Annexin V staining
followed by FACS-analysis? Wells w/o bacteria will be negative controls. You
don't need to count the cells, % IP-/AnxV+ in the sample will be the % of
dieing cells in the well every day.
<tflo at systemsbiology.org> wrote in message
news:20021118024144.27064.qmail at ww02.hostica.com...
> 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:
>> - detachment of the adherent macrophages is not an option, which precludes
> normal counting. Also, the cell growth and death is very uneven in the
wells
> and make microscopy methods difficult (biased selection of fields) - and
tedious
>> - the presence of bacteria precludes methods like MTT, WST-1 etc. due to
the
> bacterial metabolism of these substrates. Similarly, crystal violet and
neutral
> red will also stain bacteria.
>> - LDH-type assays can be used in some experiments (short-term), but as I
have
> read that the stability of LDH in tissue cultures is about 9 hours, it
will be
> difficult to monitor cell viability or cell death during a 10-day
infection
> period?? Or can I??
>> I'll be very happy if anyone could help me out here,
>> Sincere thanks,
> Trude
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