(by nobody from nospam.not)
Tue Jul 17 05:28:07 EST 2007
Kyle Legate <legatek from hotmail.com> wrote in
news:5g32olF3f0u12U1 from mid.individual.net:
> Yvonne Couch wrote:
>> Hi all,
>> I was to run a luciferase assay to see how much ATP my cells are
>> producing. For this I thought I would take an aliquot of medium from
>> each set of treated cells and use it in the Invitrogen
>> ATP-determination kit which employs luciferase.
> What makes you think your cells are secreting ATP into the medium?
> Wouldn't it make sense to make a lysate, and therefore concerns about
> medium are no longer necessary?
Some cells do secrete ATP. One method for measuring the secretion
component of platelet aactivation is the measurement of secreted ATP.
However, plasma contains nucleotidases, and the luciferase does not live
forever (it's signal degrades over time), so it is necessary to start ATP
measurement within seconds after adding a stimulus to platelets. Our lab
uses Chrono-Log equipment and reagents, so I believe that looking at
their website may help a little. In particular, the set of curves on
this page: <http://chronolog.com/CLNEWS.HTM>. The equipment is not
cheap, and really aims at platelet functionalities, rather than just ATP
Making a cell lysate has the "problem" of mixing all cellular
compartments, both those with ATP and those with nucleotidases and
phosphatases. Therefore, again time is of the essence. The luciferase
has to see the ATP before the other metabolizing enzymes, AND the
luciferase signal has to be recorded before either luciferase or other
enzymes have consumed all the ATP.
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