Quenching FITC (fwd)

Robert Preston rapr at MED.PITT.EDU
Thu Jun 16 22:04:02 EST 1994


>      The second possibility is to lower the pH of the cell solution 
> after the bacteria have been phagocytized.  The pK of fluorescein 
> is typically 6.5 and the fluorescent species is the deprotonated
> species.  So, lower pH to say 5.0 is enough to significantly 
> reduce the fluorescence.
> 
>      Mark Griep
>      mgriep at unlinfo.unl.edu
> 
> 
That's an elegant proposal, esp. if the cells can tolerate the pH 5.
Also note that the excitation peak for the acid form of fluorescein
is lower than that of the fully ionized form, so you can maiximize
the pH-dependent fluorescence change by using a narrow-band excitation
filter (10 nm bandwidth interference filter - omega optical makes good
ones) centered at around 490-495 nm.  This brings up the question,
how do you control for the fact that the FITC-labeled bacteria enter
a low pH lysosome where the fluorescence is quenched by the same 
pH-dependent mechanism?  

Rob 
rapr at med.pitt.edu





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