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Date: Thu, 16 Aug 2007 20:42:33 +0100
From: Ulrika Johansson <U.Johansson@qmul.ac.uk>
Subject: Re: CFSE compensation
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Hi Maria,

You mention that the FL1 peak is not very narrow; is this for the  
stimulated T cells? I too wonder if your customer has observed	
proliferation (if a mitogen is used it should be obvious just lookig  
down the microscope).

Just a thought, but if a high concentration of CSFE is used then I  
know from experience that it can sometimes be difficult to discern  
peaks; and if a mitogen has been used, and if your researcher is  
homing in on T cells, it's likely that all T cells have proliferated  
(all this may thus result in a broad looking sort of 'peak'.

Good luck,
BW
Ulrika


Quoting rozenkov@netscape.net:

>
> Hi Maria,
>
> It is rather difficult to make CFSE-T cells non-proliferating by   
> compensation. How does the single-parameter FL1 (FITC) histogram   
> look? Are all cells contained in the same peak as in a   
> non-stimulated control? (Seeing the pictures here would help.)
>
> I guess even if you have done 6-colour auto-comp, you can just run a	
>  CFSE-only (no antibodies) stimulated cell sample and see how it  
> looks.
>
> Another question: does you colleague see proliferation in the same   
> samples by other parameters, e.g. thymidine incorporation or at   
> least morphologically?
>
> Regards.
>
> Vladislav Rozenkov
>
>
> -----Original Message-----
> From: Maria Candela Iglesias Chiesa <candela@iner.gob.mx>
> To: cyto-inbox
> Sent: Tue, 14 Aug 2007 3:32 am
> Subject: CFSE compensation
>
>
>
>
> 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@iner.gob.mx
>
> candela.iglesias@cieni.org.mx
>
>  
>
>
>
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_______________________________
Ulrika Johansson, PhD
Department of Haematology
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e U.Johansson@qmul.ac.uk