Flow cytometry with E.coli tagged with GFP

Esa-Matti Lilius esa-matti.lilius at utu.fi
Tue Mar 12 09:05:56 EST 2002


Dear flowers,

We have tagged E.coli with GFP. When we incubate the cells in the wells of a
microplate  and measure fluorescence with  a plate reader fluorometer we
observe a certain increase in fluorescence over incubation time (4-fold). We
estimate that this increase reflects the growth of the cell population. We
see just the same increase in OD at 620 nm read with a plate reader
photometer. CFUs show the same increase, too. As well we see the same
increase with flow cytometer when we measure the number of fluorescent cells
from samples incubated identically. The problem is that the mean
fluorescence intensity of the fluorescent cells increases over the same time
period according to flow cytometer (2-fold). We anticipate that number of
fluorescent cells should be multiplied with mean fluorescence intensity to
get the total fluorescence of the cell population and the increase in the
product (2 x 4 = 8-fold) should be the same as increase in  fluorescence
intensity in fluorometer. This is, however, not the case. Have we understood
something wrong with the results obtained by flow cytometer?

Esa-Matti Lilius

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