Re: pH, fluorescein and internalization.

Hi Uriel,

Generally speaking, receptor internalization, esp. upon ligand binding, 
is a fairly rapid process that must be examined using a time course.  
The key to the assay is to fix your samples after collecting each time 
point and then re-acquire the data on the fixed cells in a neutral pH 
buffer (PBS).  If you recover the fluorescence after fixation then you 
can be confident the receptor was internalized and was sequestered into 
an acidic compartment.	The assay will differentiate between 
internalization and instances where the receptor's extracellular domain 
is cleaved from the cell surface or your probe simply falls off in 
response to treatment because you will not recover the fluorescence.  At 
longer time points (> 30 minutes) you may not recover total fluorescence 
because some protein-fluorochrome probes will be degraded.  At early 
time points (<10-15 minutes), you should recover > 90% of your 
fluorescence, relative to untreated control.  In the case of transferrin 
receptor (CD71), CD19 and MHC ClassII, incubation at 37'C is sufficient 
to see rapid internalization within 10 minutes. 

Regards, TomD, aka, Thomas Delohery, writing from home.
current position with Sanofi-Aventis, Oncology, Hematological Malignancies.


Uriel TK wrote:

> Friends,
>  
> Following the discussion on receptor internalization, I would like to 
> share with you a couple of questions, and hope you can help with 
> clarifications. 
>  
> I can understand that fluorescein can be used based on its pH 
> dependence as an EVIDENCE of internalization, by detecting the loss of 
> fluorescence (by flow or microscopy) due to acidification of 
> endosomes. But certainly it cannot be used as a MEASURE of 
> internalization, or can it? how can you control or detect the pH of 
> the vesicles given your treatment, the time elapsed, the asynchronous 
> nature of the process, the continuation of the metabolism of the 
> endocytic pathway, etc? For measuring it would have to be used 
> together with a non-pH sensitive probe, as Thomas D. mentioned. And 
> still then I think some problems would appear regarding the 
> determination of the internalized vs non-internalized fraction.
> I understand fluorescein is used as a tracer or marker of the 
> endocytic or transport pathways in a variety of ways but wouldn't 
> it be a bad choice given the expected loss of fluorescence? Mol probes 
> has an interesting twist, where highly conjugated proteins or dextran 
> cause internal quenching and after proteolysis there is dequenching, 
> but once again the pH would affect fluorescein and it would be a bad 
> choice given non-pH dependent alternatives. 
> (http://probes.invitrogen.com/handbook/sections/1407.html).
> Finally, applications where fluorescein is not used in the endocytic 
> pathway (like the FDA viability assay) should not suffer from these 
> problems, or maybe it would be more cautious to say that they probably 
> suffer to a much lesser extent from these uncertainties.
>  
> Therefore, given fluorescein's subtleties and given the availability 
> of other probes which overcome them, what escapes me, as I said in a 
> previous post, is: why is it still so widely used? Am I missing 
> something? or is it plain lack of knowledge regarding fluorescenin's 
> pH dependence? 
>  
> Thanks,
>  
> uriel.
> Uriel Trahtemberg, M.Sc.
> MD/PhD student
> The Laboratory for Cellular and Molecular Immunology
> The Hebrew University - Hadassah Medical Organization
> Jerusalem - ISRAEL
>  
> "We shall defend our island, whatever the cost may be, we shall fight 
> on the beaches, we shall fight on the landing grounds, we shall fight 
> in the fields and in the streets, we shall fight in the hills; we 
> shall never surrender."
> Winston [Leonard Spencer] Churchill