CFSE compensation

maciej simm simm at cd4cd8.com
Tue Aug 14 20:30:22 EDT 2007


Dr. Iglesias,

I'm assuming you mean "FL1" or "green fluorescence" since "FITC"  
refers to a specific analyte with different properties than CFSE  
(even though both fluoresce "green" light ;).

The stimulated/unstimulated cells may bind antibodies differently, if  
they do, they would not be a good match for compensation control...  
as per step 1 in Dr. Mario Roedere's "recipe" http://www.drmr.com/ 
compensation/Recipe.html

Do you have other CFSE users for whom the results match expectations?  
In addition to what you hear back from the list, it'd be worth it to  
talk to local users who perform similar assays and see what the  
differences are..

Maciej Simm








On Aug 13, 2007, at 10:32 AM, Maria Candela Iglesias Chiesa wrote:

> Hi everybody,
>
>
>
> A colleague is putting up a CFSE proliferation assay on T cells.  
> She hasn´t been able to see any proliferation after 6d of  
> stimulation. However, I strongly suspect this is a problem of  
> compensation more than anything else. We have a FACSAria and  
> normally do Compbeads and automatic compensation. She has run non  
> stimulated cells CFSE stained to compensate for the “FITC” channel,  
> and stained compbeads for the other channels. She hasn’t got a very  
> narrow peak in “FITC”, but the compensation works anyway. Still,  
> she doesn’t see any proliferation, and has performed her experiment  
> in a very standard way. Am I missing some obvious point in the  
> compensation procedure? How do you normally compensate when using  
> CFSE? I’ve done it manually on a 3 color expt in a Calibur and  
> never had any problem. Should I try and compensate manually even if  
> she is using 6 colors?
>
> Many thanks,
>
>
>
> Candela
>
>
>
> María Candela Iglesias, PhD
>
> Centro de Investigacion en Enfermedades Infecciosas (CIENI)
>
> Instituto Nacional de Enfermedades Respiratorias
>
> Calzada de Tlalpan 4502. Col. Seccion XVI
>
> CP 14080. Deleg. Tlalpan
>
> México DF.
>
> tel y fax +52 (55) 56 66 79 85
>
> candela at iner.gob.mx
>
> candela.iglesias at cieni.org.mx
>
>
>
>

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