>The following is a reply Bob MacDonald sent me concerning the use of a
fluorescent plate reader.
>From: macd at nwu.edu (Robert C. MacDonald)
>Subject: Re: Fluorescent plate reader
>>It will take a plate reader of unusual sensitivity if it works at all. I
>have tried but unfortunately only did the relevant calculation after buying
>the microplate fluorometer and finding it inadequate. That calculation is
>to assume that each cell expresses to a concentration of 1 micromolar (a
>very generous assumption), then calculate the mumber of molecules sensed by
>the instrument AT EACH READING. Typically, these instruments look at a
>region that is a few mm in diameter. Compare that number with the quoted
>sensitivity of the instrument. Chances are, it will not even come close.
>Bear in mind that you need a signal that is at least two orders of magnitude
> (preferably considerably more) larger than the instrument sensitivity, or
>you will not have an adequate range for the assay. My comment apply to
>looking at expressing cells growing on the plate. If one were to, say,
>remove the cells from a 6 cm dish and put them all in one well of a 96 well
>plate, it might work.
>>>>>>If anyone has any information on detecting GFP fluorescence using a
>>fluorescent plate reader could you please post it to the group generally as
>>this issue is quite important. An MSc student in our lab tried briefly to do
>>this but found that it was not sensitive enough.
>>However it may be due to the instability of the construct we were using at
>>the time rather than the sensitivity of the system so any other comments
>>would be appreciated.
>>>>Karen Scott, RRI.
>Department of Biochemistry, Molecular Biology and Cell Biology
>2153 Campus Drive,
>Evanston, IL 60208-3500
>Telephone: 847 491-5062; FAX: 847 467-1380; Email: macd at nwu.edu>>>