Fluorescent plate reader

Karen P Scott kps at rri.sari.ac.uk
Thu May 9 03:26:24 EST 1996

>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.
>Bob MacDonald
>>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.
>Robert MacDonald
>Department of Biochemistry, Molecular Biology and Cell Biology
>Northwestern University
>2153 Campus Drive,
>Evanston, IL 60208-3500
>Telephone: 847 491-5062;  FAX: 847 467-1380;  Email:  macd at nwu.edu

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