analyzing 3t3 fibroblasts

Gerstein, Rachel Rachel.Gerstein at umassmed.edu
Fri Nov 4 10:07:51 EST 2005


hi

fibroblast are much bigger and more autofluorescent, so be sure that you have transfected
controls.  If you have a good, bright reagent, you should be able to lower the PMT so
that the un-TF cells are placed in the first decade of fluor channels.

Also, I suggest using PI if you are doing live cell analysis to exclude dead cells.

Finally, people often worry alot about trypsinizing off the antigens, but if you are
careful to treat only as long as you need to and then wash thorougly, this is usually not
a problem.

good luck,
Rachel

=======================================================
Rachel M. Gerstein, Ph.D.
Assistant Professor
Department of Molecular Genetics and Microbiology
Graduate Program in Immunology/Virology
University of Massachusetts Medical School
55 Lake Avenue North
Worcester, MA 01655-0002
(508) 856-1044
(508) 856-5920 (FAX)


> ----------
> From: 	Cheng, Bertha
> Sent: 	Wednesday, November 2, 2005 4:31 PM
> To:	Cytometry Mailing List
> Subject:	analyzing 3t3 fibroblasts
> 
> Hello all,
> 
> I was given some CD40L-transfected 3T3 fibroblasts and I'd like to make sure
> they are indeed expressing CD40L.  Since our lab usually deals with blood
> samples, should the staining protocol for the fibroblasts be different than
> for PBMC? If so, what are some things I should consider to get the most
> representative data of my cells on the FACsort?  Any recommendations would
> be appreciated. Thanks
> 
> Bertha Cheng
> UCSF
> Immunogenetics and Transplantation 
> 
> 
> 




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